Role of Leaf Surface Sugars in Colonization of Plants by Bacterial Epiphytes

The relationship between nutrients leached onto the leaf surface and the colonization of plants by bacteria was studied by measuring both the abundance of simple sugars and the growth of Pseudomonas fluorescens on individual bean leaves. Data obtained in this study indicate that the population size of epiphytic bacteria on plants under environmentally favorable conditions is limited by the abundance of carbon sources on the leaf surface. Sugars were depleted during the course of bacterial colonization of the leaf surface. However, about 20% of readily utilizable sugar, such as glucose, present initially remained on fully colonized leaves. The amounts of sugars on a population of apparently identical individual bean leaves before and after microbial colonization exhibited a similar right-hand-skewed distribution and varied by about 25-fold from leaf to leaf. Total bacterial population sizes on inoculated leaves under conditions favorable for bacterial growth also varied by about 29-fold and exhibited a right-hand-skewed distribution. The amounts of sugars on leaves of different plant species were directly correlated with the maximum bacterial population sizes that could be attained on those species. The capacity of bacteria to deplete leaf surface sugars varied greatly among plant species. Plants capable of supporting high bacterial population sizes were proportionally more depleted of leaf surface nutrients than plants with low epiphytic populations. Even in species with a high epiphytic bacterial population, a substantial amount of sugar remained after bacterial colonization. It is hypothesized that residual sugars on colonized leaves may not be physically accessible to the bacteria due to limitations in wettability and/or diffusion of nutrients across the leaf surface.     

Role of leaf surface sugars in colonization of plants by bacterial epiphytes

The relationship between nutrients leached onto the leaf surface and the colonization of plants by bacteria was studied by measuring both the abundance of simple sugars and the growth of Pseudomonas fluorescens on individual bean leaves. Data obtained in this study indicate that the population size of epiphytic bacteria on plants under environmentally favorable conditions is limited by the abundance of carbon sources on the leaf surface. Sugars were depleted during the course of bacterial colonization of the leaf surface. However, about 20% of readily utilizable sugar, such as glucose, present initially remained on fully colonized leaves. The amounts of sugars on a population of apparently identical individual bean leaves before and after microbial colonization exhibited a similar right-hand-skewed distribution and varied by about 25-fold from leaf to leaf. Total bacterial population sizes on inoculated leaves under conditions favorable for bacterial growth also varied by about 29-fold and exhibited a right-hand-skewed distribution. The amounts of sugars on leaves of different plant species were directly correlated with the maximum bacterial population sizes that could be attained on those species. The capacity of bacteria to deplete leaf surface sugars varied greatly among plant species. Plants capable of supporting high bacterial population sizes were proportionally more depleted of leaf surface nutrients than plants with low epiphytic populations. Even in species with a high epiphytic bacterial population, a substantial amount of sugar remained after bacterial colonization. It is hypothesized that residual sugars on colonized leaves may not be physically accessible to the bacteria due to limitations in wettability and/or diffusion of nutrients across the leaf surface.     

Population Sizes, Immigration, and Growth of Epiphytic Bacteria on Leaves of Different Ages and Positions of Field-Grown Endive (Cichorium endivia var. latifolia)

Total, fluorescent, and pectolytic epiphytic bacterial population sizes were quantified on leaves of different age groups of broad-leaved endive during field cultivation from leaf emergence until harvest. Greater bacterial population densities (log(inf10) CFU per square centimeter) were observed on outer leaves than on inner leaves of the plants throughout the growing season. These differences were statistically significant for total bacterial populations at all sampling times and were often significant for fluorescent and pectolytic bacterial populations. At harvest, a linear gradient of decreasing densities of epiphytic bacteria from outer (older) to inner (younger) leaves of the head was significant. Leaf age influenced the frequency distribution and variability of bacterial population sizes associated with leaves of broad-leaved endive. Total bacterial population sizes were greater at leaf emergence for leaves emerging during the second half of the cultivation period than for leaves emerging earlier. The size of fluorescent and pectolytic bacterial populations on newly emerged leaves increased throughout the season as plants aged. To assess the importance of plant age on bacterial immigration at leaf emergence, bacterial densities were quantified on leaves emerging simultaneously on plants of different ages. In two of the three experiments, greater bacterial population sizes were observed on leaves emerging on younger plants. This indicates that factors other than an increase in concentration of airborne bacteria can lead to increases in population sizes at leaf emergence as plants age in the field. Results of leaf pruning experiments suggested that adjacent leaves may act as a barrier for immigration of fluorescent bacteria on newly emerged leaves. Survival of an inoculated strain of Pseudomonas fluorescens on newly emerged leaves generally did not vary with the age of plants. However, these effects were not consistent among experiments, suggesting that interactions among micro- and macroenvironmental conditions, physiological condition of leaves, and accessibility of leaves to airborne bacteria are important in controlling epiphytic bacterial population sizes.     

Note: Colonization and invasion of leaves of the aquatic macrophyteCeratophyllum demersum L. by epiphytic bacteria

The colonization of leaves of the aquatic macrophyteCeratophyllum demersum L. by epiphytic bacteria, and the hypothesis that bacterial invasion causes leaf senescence, was studied using transmission and scanning electron microscopy and light microscopy. Population densities of epiphytic bacterial communities onCeratophyllum leaves were positively correlated with leaf age. Initial settlement of bacteria on young leaves appeared to favour the boundaries between epidermal cells. On older leaves, large populations of bacteria were present over the whole surface. One third of senescentCeratophyllum leaves examined by transmission electron microscopy showed signs of bacterial invasion. Of these, up to 54% of the leaf's epidermal cells contained bacteria. Areas of cell wall degradation were associated with invasive bacteria in senescent leaves. In healthy, nonsenescent leaves, no bacterial invasion was observed. These results suggest that epiphytic bacteria did not cause leaf senescence but probably colonized the internal tissues of leaves once senescence had occurred.     

Influence of Immigration on Epiphytic Bacterial Populations on Navel Orange Leaves

Factors that influenced the increase in epiphytic bacterial population size on navel orange leaves during winter months were investigated to test the assumption that such populations were the result of multiplication on orange leaves. The population sizes of bacteria of different kinds, including ice nucleation-active (Ice(sup+)) bacteria, were from 6- to 30-fold larger on leaves of navel orange trees adjacent to other plant species than on trees growing near other citrus species. Total and Ice(sup+) bacterial population sizes on other plant species growing near navel orange trees were from 18- to 60-fold and 2- to 18,000-fold larger, respectively, than on navel orange trees. About twice the number of bacterial cells of a given type were deposited onto petri dishes opened simultaneously in navel orange orchards with other plant species nearby as in orchards surrounded by citrus trees. Epiphytic bacteria and airborne bacteria were more numerous near the upwind edge of orchards bordering on other plant species, but not in orchards adjacent to other citrus trees, and decreased with distance from other plant species. Navel orange leaves also exhibited progressive increases in the ability to supercool as a function of increasing distance from the upwind edge of orchards adjacent to other plant species but not in orchards adjacent to other citrus trees. While the population size of three different bacterial strains remained nearly constant for 60 days after inoculation, total bacterial populations increased more than 50-fold during this period. These results suggest that immigration of bacteria from plants having high epiphytic bacterial populations could account for most, if not all, of the seasonal increase in bacterial populations on navel orange leaves and have important implications for procedures to modify bacterial communities on leaves.     

The Spread of Epiphytic Populations of Xanthomonas campestris pv. vesicatoria on Pepper in the Field

The spread of the epiphytic population of Xanthomonas campestris pv. vesicatoria and the disease it causes, bacterial leaf spot, were studied in field plots of pepper near Gainesville, Florida. In the summer of 1989, the epiphytic population of X. campestris pv. vesicatoria was dispersed to the west-northwest from point sources of diseased plants. Winds from the southeast during rainstorms were essential for the spread of bacteria in the field. In the autumn of 1989, a focus of bacterial leaf spot developed naturally near the centre of the experimental plot. The epiphytic population of X, campestris pv. vesicatoria increased sharply after a 2-day rain accompanied with strong wind. The wind was believed to be responsible for the transport of bacteria to distances 32 m from the focus. Initially in both seasons, the epiphytic populations occurred as distinct gradients from the focal sources of diseased plants. These gradients flattened over time and the disease incidence increased to near 100%, The increase in the epiphytic populations of the pathogen to > 3.0 log₁₀ (cfu cm⁻²) on healthy plants away from the foci preceded disease appearance by several weeks. Applications of cupric hydroxide plus mancozeb significantly reduced the epiphytic population of X. campestris pv. vesicatoria on pepper leaves and slowed the spread of disease in the plots.     

Xanthomonas axonopodis pv. phaseoli var. fuscans Is Aggregated in Stable Biofilm Population Sizes in the Phyllosphere of Field-Grown Beans

The occurrence of “Xanthomonas axonopodis pv. phaseoli var. fuscans” (proposed name) populations as biofilms on bean leaves was investigated during three field experiments on plots established with naturally contaminated bean seeds. Behavior of aggregated versus solitary populations was determined by quantification of culturable cells in different fractions of the epiphytic population separated by particle size. X. axonopodis pv. phaseoli var. fuscans population dynamic studies confirmed an asymptomatic and epiphytic colonization of the bean phyllosphere. For all years of experiment and cultivars tested, biofilms and solitary components of the populations were always detected. Biofilm population sizes remained stable throughout the growing season (around 10⁵ CFU/g of fresh weight) while solitary population sizes were more abundant and varied with climate. According to enterobacterial repetitive intergenic consensus fingerprinting, aggregated bacterial isolates were not different from solitary isolates. In controlled conditions, application of a hydric stress resulted in a decrease of the solitary populations on the leaf surface while the biofilm fraction remained stable. Suppression of the hydric stress allowed solitary bacterial populations to increase again. Aggregation in biofilms on leaf surfaces provides protection to the bacterial cells against hydric stress.     

Novel Method for Identifying Bacterial Mutants with Reduced Epiphytic Fitness

In order to identify novel traits involved in epiphytic colonization, a technique for the rapid identification of bacterial mutants with quantitatively different population sizes in a natural habitat based on measurements of ice nucleation activity was developed. The threshold freezing temperatures of leaves harboring different numbers of cells of ice nucleation-active Pseudomonas syringae B728a differed substantially. While few leaves containing less than about 10⁶ cells per g (fresh weight) froze at assay temperatures of -2.75°C or higher, nearly all leaves froze at these temperatures when population sizes of this strain increased to about 10⁷ cells per g (fresh weight). Presumptive epiphytic fitness mutants could readily be identified as strains which initiated freezing in fewer leaves than did other strains within a given experiment. Most Tn5-induced mutants of strain B728a which conferred a low frequency of ice nucleation on inoculated bean leaves generally had a smaller population size than the parental strain at the time of the leaf freezing assay. The leaf freezing assay was capable of differentiating samples which varied by approximately three- to fivefold in mean bacterial population size.     

Xanthomonas axonopodis pv. phaseoli var. fuscans is aggregated in stable biofilm population sizes in the phyllosphere of field-grown beans

The occurrence of "Xanthomonas axonopodis pv. phaseoli var. fuscans" (proposed name) populations as biofilms on bean leaves was investigated during three field experiments on plots established with naturally contaminated bean seeds. Behavior of aggregated versus solitary populations was determined by quantification of culturable cells in different fractions of the epiphytic population separated by particle size. X. axonopodis pv. phaseoli var. fuscans population dynamic studies confirmed an asymptomatic and epiphytic colonization of the bean phyllosphere. For all years of experiment and cultivars tested, biofilms and solitary components of the populations were always detected. Biofilm population sizes remained stable throughout the growing season (around 10(5) CFU/g of fresh weight) while solitary population sizes were more abundant and varied with climate. According to enterobacterial repetitive intergenic consensus fingerprinting, aggregated bacterial isolates were not different from solitary isolates. In controlled conditions, application of a hydric stress resulted in a decrease of the solitary populations on the leaf surface while the biofilm fraction remained stable. Suppression of the hydric stress allowed solitary bacterial populations to increase again. Aggregation in biofilms on leaf surfaces provides protection to the bacterial cells against hydric stress.     

Comparison of the Phenotypes and Genotypes of Biofilm and Solitary Epiphytic Bacterial Populations on Broad-Leaved Endive

The discovery that biofilms are ubiquitous among the epiphytic microflora of leaves has prompted research about the impact of biofilms on the ecology of epiphytic microorganisms and on the efficiency of strategies to manage these populations for disease control and to ensure food safety. Biofilms are likely to influence the microenvironment and phenotype of the microorganisms they harbor. However, it is also important to determine whether there are differences in the types of bacteria within biofilms compared to those outside of biofilms so as to better target microorganisms via disease control strategies. Broad-leaved endive (Cichorium endivia var. latifolia) harbors biofilms containing fluorescent pseudomonads. These bacteria can cause considerable post-harvest losses when this plant is used for manufacturing minimally processed salads. To determine whether the population structure of the fluorescent pseudomonads in biofilms is different from that outside of biofilms on the same leaves, bacteria were isolated quantitatively from the biofilm and solitary components of the epiphytic population on leaves of field-grown broad-leaved endive. Population structure was determined in terms of taxonomic identities of the bacteria isolated, in terms of genotypic profiles, and in terms of phenotypic traits related to surface colonization and biofilm formation. The results illustrate that there are no systematic differences in the composition and structure of biofilm and solitary populations of fluorescent pseudomonads, in terms of either genotypic profiles or phenotypic profiles of the strains. However, Gram-positive bacteria tended to occur more frequently within biofilms than outside of biofilms. We suggest that leaf colonization by fluorescent pseudomonads involves a flux of cells between biofilm and solitary states. This would allow bacteria to exploit the advantages of these two types of existence; biofilms would favor resistance to stressful conditions, whereas solitary cells could foster spread of bacteria to newly colonizable sites on leaves as environmental conditions fluctuate.     

Characterization of Epiphytic Bacterial Communities from Grapes,Leaves, Bark and Soil of Grapevine Plants Grown,and Their Relations

Despite its importance in plant health and crop quality, the diversity of epiphytic bacteria on grape berries and other plant parts, like leaves and bark, remains poorly described, as does the role of telluric bacteria in plant colonization. In this study, we compare the bacterial community size and structure in vineyard soils, as well as on grapevine bark, leaves and berries. Analyses of culturable bacteria revealed differences in the size and structure of the populations in each ecosystem. The highest bacteria population counts and the greatest diversity of genera were found in soil samples, followed by bark, grapes and leaves. The identification of isolates revealed that some genera – Pseudomonas, Curtobacterium, and Bacillus – were present in all ecosystems, but in different amounts, while others were ecosystem-specific. About 50% of the genera were common to soil and bark, but absent from leaves and grapes. The opposite was also observed: grape and leaf samples presented 50% of genera in common that were absent from trunk and soil. The bacterial community structure analyzed by T-RFLP indicated similarities between the profiles of leaves and grapes, on the one hand, and bark and soil, on the other, reflecting the number of shared T-RFs. The results suggest an interaction between telluric bacterial communities and the epiphytic bacteria present on the different grapevine parts.     

B. thuringiensis is a Poor Colonist of Leaf Surfaces

The ability of several Bacillus thuringiensis strains to colonize plant surfaces was assessed and compared with that of more common epiphytic bacteria. While all B. thuringiensis strains multiplied to some extent after inoculation on bean plants, their maximum epiphytic population sizes of 10⁶ cfu/g of leaf were always much less than that achieved by other resident epiphytic bacteria or an epiphytically fit Pseudomonas fluorescens strain, which attained population sizes of about 10⁷ cfu/g of leaf. However B. thuringiensis strains exhibited much less decline in culturable populations upon imposition of desiccation stress than did other resident bacteria or an inoculated P. fluorescens strain, and most cells were in a spore form soon after inoculation onto plants. B. thuringiensis strains produced commercially for insect control were not less epiphytically fit than strains recently isolated from leaf surfaces. The growth of B. thuringiensis was not affected by the presence of Pseudomonas syringae when co-inoculated, and vice versa. B. thuringiensis strains harboring a green fluorescent protein marker gene did not form large cell aggregates, were not associated with other epiphytic bacteria, and were not found associated with leaf structures, such as stomata, trichomes, or veins when directly observed on bean leaves by epifluorescent microscopy. Thus, B. thuringiensis appears unable to grow extensively on leaves and its common isolation from plants may reflect immigration from more abundant reservoirs elsewhere.     

Cultivable Bacteria Populations Associated with Leaves of Banana and Plantain Plants and Their Antagonistic Activity Against Mycosphaerella fijiensis

Mycosphaerella fijiensis is the etiological agent of Black Sigatoka, a fungal disease that affects production of banana and plantain crops in tropical regions. The sizes of cultivable epiphytic and endophytic bacterial populations, aerobic endospore forming bacteria (AEFB), and antagonist bacteria against M. fijiensis isolated from three Musa spp. cultivars from Urabá (Colombia) were studied, in order to find a suitable screening strategy to isolate antagonistic bacteria. Most of the variability found in the epiphytic and endophytic bacterial community sizes among fruit trees was explained by the cultivar differences. We found population sizes ranging from 1.25?×?10(3) to 9.64?×?10(5)?CFU/g of fresh leaf and found that 44?% of total cultivable bacteria belong to the AEFB group. We isolated 648 AEFB from three different cultivars and assessed their antagonistic activity against M. fijiensis using the cell-free supernatant obtained from bacterial liquid cultures in three different in vitro assays. Five percent of those bacteria showed higher percent inhibition than the positive control Bacillus subtilis UA321 has (percent inhibition?=?84?±?5) in the screening phase. Therefore, they were selected as antagonistic bacteria against the pathogen. The strains with the highest percentage of antagonism were found in older leaves for the three cultivars, given support to recommend this group of leaves for future samplings. Some of these isolated bacteria affected the mycelium and ascospores morphology of the fungus. They also presented in vitro characteristics related to a successful colonization of the phylloplane such as indolic compounds, surfactant production, and biofilm formation, which makes them possible, potential candidates as biological control agents.     

Differences between Pseudomonas syringae pv. syringae B728a and Pantoea agglomerans BRT98 in epiphytic and endophytic colonization of leaves

The leaf colonization strategies of two bacterial strains were investigated. The foliar pathogen Pseudomonas syringae pv. syringae strain B728a and the nonpathogen Pantoea agglomerans strain BRT98 were marked with a green fluorescent protein, and surface (epiphytic) and subsurface (endophytic) sites of bean and maize leaves in the laboratory and the field were monitored to see if populations of these strains developed. The populations were monitored using both fluorescence microscopy and counts of culturable cells recovered from nonsterilized and surface-sterilized leaves. The P. agglomerans strain exclusively colonized epiphytic sites on the two plant species. Under favorable conditions, the P. agglomerans strain formed aggregates that often extended over multiple epidermal cells. The P. syringae pv. syringae strain established epiphytic and endophytic populations on asymptomatic leaves of the two plant species in the field, with most of the P. syringae pv. syringae B728a cells remaining in epiphytic sites of the maize leaves and an increasing number occupying endophytic sites of the bean leaves in the 15-day monitoring period. The epiphytic P. syringae pv. syringae B728a populations appeared to originate primarily from multiplication in surface sites rather than from the movement of cells from subsurface to surface sites. The endophytic P. syringae pv. syringae B728a populations appeared to originate primarily from inward movement through the stomata, with higher levels of multiplication occurring in bean than in maize. A rainstorm involving a high raindrop momentum was associated with rapid growth of the P. agglomerans strain on both plant species and with rapid growth of both the epiphytic and endophytic populations of the P. syringae pv. syringae strain on bean but not with growth of the P. syringae pv. syringae strain on maize. These results demonstrate that the two bacterial strains employed distinct colonization strategies and that the epiphytic and endophytic population dynamics of the pathogenic P. syringae pv. syringae strain were dependent on the plant species, whereas those of the nonpathogenic P. agglomerans strain were not.     

Variance in composition of inquiline communities in leaves of Sarracenia purpurea L. on multiple spatial scales

A survey of the abundances of species that inhabit the water-bearing leaves of the pitcher plant Sarracenia purpurea was conducted at several different spatial scales in northern Florida. Individual leaves are hosts to communities of inquiline species, including mosquitoes, midges, mites, copepods, cladocerans, and a diverse bacterial assemblage. Inquiline communities were quantified from four pitchers per plant, three plants per subpopulation, two subpopulations per population, and three populations. Species varied in abundance at different spatial scales. Variation in the abundances of mosquitoes and copepods was not significantly associated with any spatial scale. Midges varied in abundance at the level of populations; one population contained significantly more midges than the other two. Cladocerans varied at the level of the subpopulation, whereas mites varied at the level of the individual plants. Bacterial communities were described by means of Biolog plates, which quantify the types of carbon media used by the bacteria in each pitcher. Bacterial communities were found to vary significantly in composition among individual plants but not among populations or subpopulations. These results suggest that independent factors determining the abundances of individual species are important in determining community patterns in pitcher-plant inquilines.     

Ice nucleation temperature of individual leaves in relation to population sizes of ice nucleation active bacteria and frost injury

Ice nucleation temperatures of individual leaves were determined by a tube nucleation test. With this assay, a direct quantitative relationship was obtained between the temperatures at which ice nucleation occurred on individual oat (Avena sativa L.) leaves and the population sizes of ice nucleation active (INA) bacteria present on those leaves. In the absence of INA bacteria, nucleation of supercooled growth-chamber grown oat leaves did not occur until temperatures were below approximately −5°C. Both nucleation temperature and population size of INA bacteria were determined on the same individual, field-grown oat leaves. Leaves with higher ice nucleation temperatures harbored larger populations of INA bacteria than did leaves with lower nucleation temperatures. Log₁₀ mean populations of INA bacteria per leaf were 5.14 and 3.51 for leaves with nucleation temperatures of −2.5°C and −3.0°C, respectively. Nucleation frequencies (the ratio of ice nuclei to viable cells) of INA bacteria on leaves were lognormally distributed. Strains from two very different collections of Pseudomonas syringae and one of Erwinia herbicola were cultured on nutrient glycerol agar and tested for nucleation frequency at −5°C. Nucleation frequencies of these bacterial strains were also lognormally distributed within each of the three sets. The tube nucleation test was used to determine the frequency with which individual leaves in an oat canopy harbored large populations of INA bacteria throughout the growing season. This test also predicted relative frost hazard to tomato (Lycopersicon esculentum Mill) plants.     

Differences between Pseudomonas syringae pv. syringae B728a and Pantoea agglomerans BRT98 in Epiphytic and Endophytic Colonization of Leaves

The leaf colonization strategies of two bacterial strains were investigated. The foliar pathogen Pseudomonas syringae pv. syringae strain B728a and the nonpathogen Pantoea agglomerans strain BRT98 were marked with a green fluorescent protein, and surface (epiphytic) and subsurface (endophytic) sites of bean and maize leaves in the laboratory and the field were monitored to see if populations of these strains developed. The populations were monitored using both fluorescence microscopy and counts of culturable cells recovered from nonsterilized and surface-sterilized leaves. The P. agglomerans strain exclusively colonized epiphytic sites on the two plant species. Under favorable conditions, the P. agglomerans strain formed aggregates that often extended over multiple epidermal cells. The P. syringae pv. syringae strain established epiphytic and endophytic populations on asymptomatic leaves of the two plant species in the field, with most of the P. syringae pv. syringae B728a cells remaining in epiphytic sites of the maize leaves and an increasing number occupying endophytic sites of the bean leaves in the 15-day monitoring period. The epiphytic P. syringae pv. syringae B728a populations appeared to originate primarily from multiplication in surface sites rather than from the movement of cells from subsurface to surface sites. The endophytic P. syringae pv. syringae B728a populations appeared to originate primarily from inward movement through the stomata, with higher levels of multiplication occurring in bean than in maize. A rainstorm involving a high raindrop momentum was associated with rapid growth of the P. agglomerans strain on both plant species and with rapid growth of both the epiphytic and endophytic populations of the P. syringae pv. syringae strain on bean but not with growth of the P. syringae pv. syringae strain on maize. These results demonstrate that the two bacterial strains employed distinct colonization strategies and that the epiphytic and endophytic population dynamics of the pathogenic P. syringae pv. syringae strain were dependent on the plant species, whereas those of the nonpathogenic P. agglomerans strain were not.     

Occurrence and ultrastructural characterization of bacteria in association with and isolated from Azolla caroliniana.

The occurrence and ultrastructure of bacteria in leaf cavities of symbiotic Azolla caroliniana were examined by transmission electron microscopy. Bacteria were observed in all leaf cavities of Azolla cultures. Five ultrastructurally distinct types of bacteria were observed in each individual leaf cavity. Features used to characterize the bacteria included morphology, cell wall structure, and cytoplasmic organization. At least one gram-positive and as many as four gram-negative types of bacteria reside in leaf cavities of A. caroliniana. The morphological and ultrastructural characteristics of the gram-positive bacterium suggest that it is an Arthrobacter sp. The gram-negative bacteria could not be cultured; therefore, they have not been classified further. Bacterial cell shape and cell wall structure were similar in leaf cavities of different ages, but cell size and cytoplasmic composition varied. The relative contributions of each bacterial type to the total community within individual leaves was determined. Ultrastructural characteristics of bacterial isolates cultured from A. caroliniana in a free-living state were also examined.     

Loline alkaloid production by fungal endophytes of Fescue species select for particular epiphytic bacterial microflora

The leaves of fescue grasses are protected from herbivores by the production of loline alkaloids by the mutualist fungal endophytes Neotyphodium sp. or Epichloë sp. Most bacteria that reside on the leaf surface of such grasses can consume these defensive chemicals. Loline-consuming bacteria are rare on the leaves of other plant species. Several bacterial species including Burkholderia ambifaria recovered from tall fescue could use N-formyl loline as a sole carbon and nitrogen source in culture and achieved population sizes that were about eightfold higher when inoculated onto plants harboring loline-producing fungal endophytes than on plants lacking such endophytes or which were colonized by fungal variants incapable of loline production. In contrast, mutants of B. ambifaria and other bacterial species incapable of loline catabolism achieved similarly low population sizes on tall fescue colonized by loline-producing Neotyphodium sp. and on plants lacking this endophytic fungus. Lolines that are released onto the surface of plants benefiting from a fungal mutualism thus appear to be a major resource that can be exploited by epiphytic bacteria, thereby driving the establishment of a characteristic bacterial community on such plants.     

The Role of Pigmentation, Ultraviolet Radiation Tolerance, and Leaf Colonization Strategies in the Epiphytic Survival of Phyllosphere Bacteria

Phenotypic mechanisms that enhance bacterial UVR survival typically include pigmentation and DNA repair mechanisms which provide protection from UVA and UVB wavelengths, respectively. In this study, we examined the contribution of pigmentation to field survival in Clavibacter michiganensis and evaluated differences in population dynamics and leaf colonization strategies. Two C. michiganensis pigment-deficient mutants were significantly reduced in UVA radiation survival in vitro; one of these mutants also exhibited reduced field populations on peanut when compared to the wild-type strain over the course of replicate 25-day experiments. The UVR-tolerant C. michiganensis strains G7.1 and G11.1 maintained larger epiphytic field populations on peanut compared to the UVR-sensitive C. michiganensis T5.1. Epiphytic field populations of C. michiganensis utilized the strategy of solar UVR avoidance during leaf colonization resulting in increased strain survival on leaves after UVC irradiation. These results further demonstrate the importance of UVR tolerance in the ability of bacterial strains to maintain population size in the phyllosphere. However, an examination of several bacterial species from the peanut phyllosphere and a collection of environmental Pseudomonas spp. revealed that sensitivity to UVA and UVC radiation was correlated in some but not all of these bacteria. These results underscore a need to further understand the biological effects of different solar wavelength groups on microbial ecology.