Comparison of the Behavior of Epiphytic Fitness Mutants of Pseudomonas syringae under Controlled and Field Conditions

The epiphytic fitness of four Tn5 mutants of Pseudomonas syringae that exhibited reduced epiphytic fitness in the laboratory was evaluated under field conditions. The mutants differed more from the parental strain under field conditions than under laboratory conditions in their survival immediately following inoculation onto bean leaves and in the size of the epiphytic populations that they established, demonstrating that their fitness was reduced more under field conditions than in the laboratory. Under both conditions, the four mutants exhibited distinctive behaviors. One mutant exhibited particularly large population decreases and short half-lives following inoculation but grew epiphytically at near-wild-type rates, while the others exhibited reduced survival only in the warmest, driest conditions tested and grew epiphytically at reduced rates or, in the case of one mutant, not at all. The presence of the parental strain, B728a, did not influence the survival or growth of three of the mutants under field conditions; however, one mutant, an auxotroph, established larger populations in the presence of B728a than in its absence, possibly because of cross-feeding by B728a in planta. Experiments with B728a demonstrated that established epiphytic populations survived exposure of leaves to dry conditions better than newly inoculated cells did and that epiphytic survival was not dependent on the cell density in the inoculum. Three of the mutants behaved similarly to two nonpathogenic strains of P. syringae, suggesting that the mutants may be altered in traits that are missing or poorly expressed in naturally occurring nonpathogenic epiphytes.     

Characteristics of Insertional Mutants of Pseudomonas syringae with Reduced Epiphytic Fitness

Random Tn5 mutagenesis was used to identify genes ir. Pseudomonas syringae which contribute to epiphytic fitness. Mutants were selected on the basis of deficiencies in epiphytic growth or survival on plants rather than deficiencies in predetermined phenotypes exhibited in culture. A sample freezing procedure was used to measure the population sizes of 5,300 mutants of P. syringae exposed to alternating wet and dry conditions on bean leaves in growth chambers. Eighty-two mutants exhibited reduced population sizes. Of these mutants, over half exhibited a reduced ability to survive the stresses associated with dry leaves, while others grew more slowly or attained reduced stationary-phase population sizes on leaves. While some epiphytic fitness mutants were altered in phenotypes that could be measured in culture, many mutants were not altered in any in vitro phenotype examined. Only three of the epiphytic fitness mutants were auxotrophs, and none had catabolic deficiencies for any of 31 organic compounds tested. Other mutants that exhibited reductions in one or more of the following were identified: motility, osmotolerance, desiccation tolerance, growth rate in batch culture, and extracellular polysaccharide production. All of the mutants retained the abilities to produce disease symptoms on the compatible host plant, bean, to incite a hypersensitive response on the non-host plant, tobacco, and to produce a fluorescent pyoverdine siderophore.     

Quorum sensing regulates exopolysaccharide production, motility, and virulence in Pseudomonas syringae

The N-acyl homoserine lactone (AHL)-mediated quorum-sensing system in the phytopathogen Pseudomonas syringae pv. syringae requires the AHL synthase AhlI and the regulator AhlR, and is additionally subject to regulation by AefR. The contribution of quorum sensing to the expression of a variety of traits expected to be involved in epiphytic fitness and virulence of P syringae were examined. Both an aefR- mutant and an ahlI- ahlR- double mutant, deficient in AHL production, were significantly impaired in alginate production and had an increased susceptibility to hydrogen peroxide compared with the wild-type strain. These mutants were hypermotile in culture, invaded leaves more rapidly, and caused an increased incidence of brown spot lesions on bean leaves after a 48-h moist incubation. Interestingly, an aefR- mutant was both the most motile and virulent. Like the wild-type strain, the AHL-deficient mutant strains incited water-soaked lesions on bean pods. However, lesions caused by an ahlI- ahlR- double mutant were larger, whereas those incited by an aefR- mutant were smaller. In contrast, tissue maceration of pods, which occurs at a later stage of infection, was completely abolished in the AHL-deficient mutants. Both the incidence of disease and in planta growth of P syringae pv. tabaci were greatly reduced in transgenic tobacco plants that produced AHL compared with wild-type plants. These results demonstrate that quorum sensing in E syringae regulates traits that contribute to epiphytic fitness as well as to distinct stages of disease development during plant infection.     

Flagellar Motility Confers Epiphytic Fitness Advantages upon Pseudomonas syringae

The role of flagellar motility in determining the epiphytic fitness of an ice-nucleation-active strain of Pseudomonas syringae was examined. The loss of flagellar motility reduced the epiphytic fitness of a normally motile P. syringae strain as measured by its growth, survival, and competitive ability on bean leaf surfaces. Equal population sizes of motile parental or nonmotile mutant P. syringae strains were maintained on bean plants for at least 5 days following the inoculation of fully expanded primary leaves. However, when bean seedlings were inoculated before the primary leaves had expanded and bacterial populations on these leaves were quantified at full expansion, the population size of the nonmotile derivative strain reached only 0.9% that of either the motile parental or revertant strain. When fully expanded bean primary leaves were coinoculated with equal numbers of motile and nonmotile cells, the population size of a nonmotile derivative strain was one-third of that of the motile parental or revertant strain after 8 days. Motile and nonmotile cells were exposed in vitro and on plants to UV radiation and desiccating conditions. The motile and nonmotile strains exhibited equal resistance to both stresses in vitro. However, the population size of a nonmotile strain on leaves was less than 20% that of a motile revertant strain when sampled immediately after UV irradiation. Epiphytic populations of both motile and nonmotile P. syringae declined under desiccating conditions on plants, and after 8 days, the population size of a nonmotile strain was less than one-third that of the motile parental or revertant strain.     

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.     

Contribution of Indole-3-Acetic Acid Production to the Epiphytic Fitness of Erwinia herbicola

Erwinia herbicola 299R produces large quantities of indole-3-acetic acid (IAA) in culture media supplemented with l-tryptophan. To assess the contribution of IAA production to epiphytic fitness, the population dynamics of the wild-type strain and an IAA-deficient mutant of this strain on leaves were studied. Strain 299XYLE, an isogenic IAA-deficient mutant of strain 299R, was constructed by insertional interruption of the indolepyruvate decarboxylase gene of strain 299R with the xylE gene, which encodes a 2,3-catechol dioxygenase from Pseudomonas putida mt-2. The xylE gene provided a useful marker for monitoring populations of the IAA-deficient mutant strain in mixed populations with the parental strain in ecological studies. A root bioassay for IAA, in which strain 299XYLE inhibited significantly less root elongation than strain 299R, provided evidence that E. herbicola produces IAA on plant surfaces in amounts sufficient to affect the physiology of its host and that IAA production in strain 299R is not solely an in vitro phenomenon. The epiphytic fitness of strains 299R and 299XYLE was evaluated in greenhouse and field studies by analysis of changes in the ratio of the population sizes of these two strains after inoculation as mixtures onto plants. Populations of the parental strain increased to approximately twice those of the IAA-deficient mutant strain after coinoculation in a proportion of 1:1 onto bean plants in the greenhouse and onto pear flowers in field studies. In all experiments, the ratio of the population sizes of strain 299R and 299XYLE increased during periods of active growth on plant tissue but not when population sizes were not increasing with time.

Many plant-associated bacteria have the ability to produce the plant growth regulator indole-3-acetic acid (IAA) (5, 9, 25, 33). IAA is involved in diseases caused by gall- and knot-forming bacterial species (33); however, its role in other bacteria remains undefined. It is unclear whether these bacteria produce IAA during colonization of plant surfaces and whether this metabolite is beneficial to the bacteria during their growth and survival in the phyllosphere. The production of IAA may enable bacteria to detoxify tryptophan analogues present on plant surfaces (15), to downregulate genes involved in plant defense responses (33), or to inhibit the development of the hypersensitive response by plants (26). We recently demonstrated that the ipdC gene, which encodes the indolepyruvate decarboxylase of Erwinia herbicola (Pantoea agglomerans) 299R and which is involved in the indolepyruvate pathway for IAA synthesis in this epiphytic strain (2), is osmoresponsive and plant inducible (3). We hypothesized that the secretion of IAA may modify the microhabitat of epiphytic bacteria by increasing nutrient leakage from plant cells; enhanced nutrient availability may better enable IAA-producing bacteria to colonize the phyllosphere and may contribute to their epiphytic fitness (1).Few studies have attempted to determine the ecological significance of IAA production in pathogenic bacteria. Varvaro and Martella (31) showed that IAA-deficient mutants of Pseudomonas syringae pv. savastanoi, obtained by selection for resistance to α-methyltryptophan, were reduced in their ability to colonize and survive on olive leaf surfaces. The survival of an α-methyltryptophan-resistant IAA-deficient mutant of P. syringae pv. savastanoi in knots also was affected, its population declining more rapidly than that of the parental strain when inoculated alone into oleander leaf tissue (28). The importance of IAA production in bacterial colonization of bean leaves was also tested with the brown spot pathogen P. syringae pv. syringae and an IAA-deficient mutant derived by insertional mutagenesis (21). Although no difference in the survival of the parental and mutant strains on bean leaves was observed in the greenhouse, a small difference in their behavior was apparent in experiments conducted in a mist chamber (21). There have been no studies of the role of IAA production in plant-associated bacteria that do not cause disease.IAA biosynthesis is not essential for bacterial growth and survival, since IAA-deficient mutants grow as well as their IAA-producing parental strain in vitro (2, 29). Large differences in the epiphytic behaviors of IAA-producing bacteria and isogenic IAA-deficient mutants consequently would not be expected. Even small contributions of IAA production to epiphytic fitness could account for the common presence of this phenotype in epiphytic bacteria (19). Measurements of changes in the ratio of two strains following coinoculation, a common approach in ecological studies, can allow the detection of even small differences in the competitive behaviors of two organisms. This approach can detect much smaller differences in behavior between closely related species than comparison of populations of these species when present singly in separate habitats (16). In this study, we tested the role of IAA in the epiphytic fitness of E. herbicola by comparing the relative changes in the population sizes of the parental and IAA-deficient mutant strains with time after their inoculation onto plants in both controlled and field environments.     

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.     

Superoxide dismutase activity in Pseudomonas putida affects utilization of sugars and growth on root surfaces

To investigate the role of superoxide dismutases (SOD) in root colonization and oxidative stress, mutants of Pseudomonas putida lacking manganese-superoxide dismutase (MnSOD) (sodA), iron-superoxide dismutase (FeSOD) (sodB), or both were generated. The sodA sodB mutant did not grow on components washed from bean root surfaces or glucose in minimal medium. The sodB and sodA sodB mutants were more sensitive than wild type to oxidative stress generated within the cell by paraquat treatment. In single inoculation of SOD mutants on bean, only the sodA sodB double mutant was impaired in growth on root surfaces. In mixed inoculations with wild type, populations of the sodA mutant were equal to those of the wild type, but levels of the sodB mutant and, to a great extent, the sodA sodB mutant, were reduced. Confocal microscopy of young bean roots inoculated with green fluorescent protein-tagged cells showed that wild type and SOD single mutants colonized well predominantly at the root tip but that the sodA sodB double mutant grew poorly at the tip. Our results indicate that FeSOD in P. putida is more important than MnSOD in aerobic metabolism and oxidative stress. Inhibition of key metabolic enzymes by increased levels of superoxide anion may cause the impaired growth of SOD mutants in vitro and in planta.     

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.     

Involvement of the exopolysaccharide alginate in the virulence and epiphytic fitness of Pseudomonas syringae pv. syringae

Alginate, a co-polymer of O-acetylated beta-1,4-linked D-mannuronic acid and L-guluronic acid, has been reported to function in the virulence of Pseudomonas syringae, although genetic studies to test this hypothesis have not been undertaken previously. In the present study, we used a genetic approach to evaluate the role of alginate in the pathogenicity of P. syringae pv. syringae 3525, which causes bacterial brown spot on beans. Alginate biosynthesis in strain 3525 was disrupted by recombining Tn5 into algL, which encodes alginate lyase, resulting in 3525.L. Alginate production in 3525.L was restored by the introduction of pSK2 or pAD4033, which contain the alginate biosynthetic gene cluster from P. syringae pv. syringae FF5 or the algA gene from P. aeruginosa respectively. The role of alginate in the epiphytic fitness of strain 3525 was assessed by monitoring the populations of 3525 and 3525.L on tomato, which is not a host for this pathogen. The mutant 3525.L was significantly impaired in its ability to colonize tomato leaves compared with 3525, indicating that alginate functions in the survival of strain 3525 on leaf surfaces. The contribution of alginate to the virulence of strain 3525 was evaluated by comparing the population dynamics and symptom development of 3525 and 3525.L in bean leaves. Although 3525. L retained the ability to form lesions on bean leaves, symptoms were less severe, and the population was significantly reduced in comparison with 3525. These results indicate that alginate contributes to the virulence of P. syringae pv. syringae 3525, perhaps by facilitating colonization or dissemination of the bacterium in planta.     

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.     

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.     

Superoxide Dismutase Activity in Pseudomonas putida Affects Utilization of Sugars and Growth on Root Surfaces

To investigate the role of superoxide dismutases (SOD) in root colonization and oxidative stress, mutants of Pseudomonas putida lacking manganese-superoxide dismutase (MnSOD) (sodA), iron-superoxide dismutase (FeSOD) (sodB), or both were generated. The sodA sodB mutant did not grow on components washed from bean root surfaces or glucose in minimal medium. The sodB and sodA sodB mutants were more sensitive than wild type to oxidative stress generated within the cell by paraquat treatment. In single inoculation of SOD mutants on bean, only the sodA sodB double mutant was impaired in growth on root surfaces. In mixed inoculations with wild type, populations of the sodA mutant were equal to those of the wild type, but levels of the sodB mutant and, to a great extent, the sodA sodB mutant, were reduced. Confocal microscopy of young bean roots inoculated with green fluorescent protein-tagged cells showed that wild type and SOD single mutants colonized well predominantly at the root tip but that the sodA sodB double mutant grew poorly at the tip. Our results indicate that FeSOD in P. putida is more important than MnSOD in aerobic metabolism and oxidative stress. Inhibition of key metabolic enzymes by increased levels of superoxide anion may cause the impaired growth of SOD mutants in vitro and in planta.     

Contribution of Nitrate Assimilation to the Fitness of Pseudomonas syringae pv. syringae B728a on Plants

The ability of Pseudomonas syringae pv. syringae to use nitrate as a nitrogen source in culture and on leaves was assessed. Substantial amounts of leaf surface nitrate were detected directly and by use of a bioreporter of nitrate on bean plants grown with a variety of nitrogen sources. While a nitrate reductase mutant, P. syringae ΔnasB, exhibited greatly reduced growth in culture with nitrate as the sole nitrogen source, it exhibited population sizes similar to those of the wild-type strain on leaves. However, the growth of the ΔnasB mutant was much less than that of the wild-type strain when cultured in bean leaf washings supplemented with glucose, suggesting that P. syringae experiences primarily carbon-limited and only secondarily nitrogen-limited growth on bean leaves. Only a small proportion of the cells of a green fluorescent protein (GFP)-based P. syringae nitrate reductase bioreporter, LK2(pOTNas4), exhibited fluorescence on leaves. This suggests that only a subset of cells experience high nitrate levels or that nitrate assimilation is repressed by the presence of ammonium or other nitrogenous compounds in many leaf locations. While only a subpopulation of P. syringae consumes nitrate at a given time on the leaves, the ability of those cells to consume this resource would be strongly beneficial to those cells, especially in environments in which nitrate is the most abundant form of nitrogen.     

Biofilm formation, epiphytic fitness, and canker development in Xanthomonas axonopodis pv. citri

The phytopathogenic bacterium Xanthomonas axonopodis pv. citri is responsible for the canker disease affecting citrus plants throughout the world. Here, we have evaluated the role of bacterial attachment and biofilm formation in leaf colonization during canker development on lemon leaves. Crystal violet staining and confocal laser scanning microscopy analysis of X. axonopodis pv. citri strains expressing the green fluorescent protein were used to evaluate attachment and biofilm formation on abiotic and biotic (leaf) surfaces. Wild-type X. axonopodis pv. citri attached to and formed a complex, structured biofilm on glass in minimal medium containing glucose. Similar attachment and structured biofilm formation also were seen on lemon leaves. An X. axonopodis pv. citri gumB mutant strain, defective in production of the extracellular polysaccharide xanthan, did not form a structured biofilm on either abiotic or biotic surfaces. In addition, the X. axonopodis pv. citri gumB showed reduced growth and survival on leaf surfaces and reduced disease symptoms. These findings suggest an important role for formation of biofilms in the epiphytic survival of X. axonopodis pv. citri prior to development of canker disease.     

Isolation and characterization of mutants of common ice plant deficient in crassulacean acid metabolism

Crassulacean acid metabolism (CAM) is a specialized mode of photosynthesis that improves water use efficiency by shifting part or all of net atmospheric CO2 uptake to the night. Genetic dissection of regulatory and metabolic attributes of CAM has been limited by the difficulty of identifying a reliable phenotype for mutant screening. We developed a novel and simple colorimetric assay to measure leaf pH to screen fast neutron-mutagenized populations of common ice plant (Mesembryanthemum crystallinum), a facultative CAM species, to detect CAM-deficient mutants with limited nocturnal acidification. The isolated CAM-deficient mutants showed negligible net dark CO2 uptake compared with wild-type plants following the imposition of salinity stress. The mutants and wild-type plants accumulated nearly comparable levels of sodium in leaves, but the mutants grew more slowly than the wild-type plants. The mutants also had substantially reduced seed set and seed weight relative to wild type under salinity stress. Carbon-isotope ratios of seed collected from 4-month-old plants indicated that C3 photosynthesis made a greater contribution to seed production in mutants compared to wild type. The CAM-deficient mutants were deficient in leaf starch and lacked plastidic phosphoglucomutase, an enzyme critical for gluconeogenesis and starch formation, resulting in substrate limitation of nocturnal C4 acid formation. The restoration of nocturnal acidification by feeding detached leaves of salt-stressed mutants with glucose or sucrose supported this defect and served to illustrate the flexibility of CAM. The CAM-deficient mutants described here constitute important models for exploring regulatory features and metabolic consequences of CAM.