Conditional survival as a selection strategy to identify plant-inducible genes of Pseudomonas syringae

A novel strategy termed habitat-inducible rescue of survival (HIRS) was developed to identify genes of Pseudomonas syringae that are induced during growth on bean leaves. This strategy is based on the complementation of metXW, two cotranscribed genes that are necessary for methionine biosynthesis and required for survival of P. syringae on bean leaves exposed to conditions of low humidity. We constructed a promoter trap vector, pTrap, containing a promoterless version of the wild-type P. syringae metXW genes. Only with an active promoter fused to metXW on pTrap did this plasmid restore methionine prototrophy to the P. syringae metXW mutant B7MX89 and survival of this strain on bean leaves. To test this method, a partial library of P. syringae genomic DNA was constructed in pTrap and a total of 1,400 B7MX89 pTrap clones were subjected to HIRS selection on bean leaves. This resulted in the enrichment of five clones, each with a unique RsaI restriction pattern of their DNA insert. Sequence analysis of these clones revealed those P. syringae genes for which putative plant-inducible activity could be assigned. Promoter activity experiments with a gfp reporter gene revealed that these plant-inducible gene promoters had very low levels of expression in minimal medium. Based on green fluorescent protein fluorescence levels, it appears that many P. syringae genes have relatively low expression levels and that the metXW HIRS strategy is a sensitive method to detect weakly expressed P. syringae genes that are active on plants. Furthermore, we found that protected sites on the leaf surface provided a higher level of enrichment for P. syringae expressing metXW than exposed sites. Thus, the metXW HIRS strategy should lead to the identification of P. syringae genes that are expressed primarily in these areas on the leaf.     

Conditional Survival as a Selection Strategy To Identify Plant-Inducible Genes of Pseudomonas syringae

A novel strategy termed habitat-inducible rescue of survival (HIRS) was developed to identify genes of Pseudomonas syringae that are induced during growth on bean leaves. This strategy is based on the complementation of metXW, two cotranscribed genes that are necessary for methionine biosynthesis and required for survival of P. syringae on bean leaves exposed to conditions of low humidity. We constructed a promoter trap vector, pTrap, containing a promoterless version of the wild-type P. syringae metXW genes. Only with an active promoter fused to metXW on pTrap did this plasmid restore methionine prototrophy to the P. syringae metXW mutant B7MX89 and survival of this strain on bean leaves. To test this method, a partial library of P. syringae genomic DNA was constructed in pTrap and a total of 1,400 B7MX89 pTrap clones were subjected to HIRS selection on bean leaves. This resulted in the enrichment of five clones, each with a unique RsaI restriction pattern of their DNA insert. Sequence analysis of these clones revealed those P. syringae genes for which putative plant-inducible activity could be assigned. Promoter activity experiments with a gfp reporter gene revealed that these plant-inducible gene promoters had very low levels of expression in minimal medium. Based on green fluorescent protein fluorescence levels, it appears that many P. syringae genes have relatively low expression levels and that the metXW HIRS strategy is a sensitive method to detect weakly expressed P. syringae genes that are active on plants. Furthermore, we found that protected sites on the leaf surface provided a higher level of enrichment for P. syringae expressing metXW than exposed sites. Thus, the metXW HIRS strategy should lead to the identification of P. syringae genes that are expressed primarily in these areas on the leaf.     

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.     

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.     

Differential expression of genes of Pseudomonas syringae on leaves and in culture evaluated with random genomic lux fusions

Differential expression of genes of Pseudomonas syringae strain B728a on plants and in culture was assessed by measuring light production by a large collection of mutant strains containing random genomic insertions of a promoterless lux operon. Reporter gene fusions were made using Tn4431 containing lux CDABE from Vibrio fisheri. Light production reproducibly increased seven-fold when n-decanal was added to cells harvested from plant surfaces, to over 800-fold when added to cells cultured on a solidified culture medium, thus increasing the sensitivity of this reporter gene system. One of the 173 mutants tested exhibited significantly higher light production on plants than on solidified culture media compared to other mutants, while one lux fusion-containing strain produced significantly more light on culture media than on plants relative to the other mutants. The plant-inducible genes identified were not required for pathogenicity of this strain. Approximately 2% of the genes of P. syringae are apparently transcribed more actively in cells growing epiphytically on plants than in common culture media indicating that bacterial cells on plants may have substantially different behaviours than that of cultured cells.     

Effect of phenotypic plasticity on epiphytic survival and colonization by Pseudomonas syringae.

The bacterial epiphyte Pseudomonas syringae MF714R was cultured on agar or in broth or collected from colonized leaves; it was then inoculated onto greenhouse-grown bean plants incubated in a growth chamber at low relative humidity or in the field or onto field-grown bean plants. Cells cultured in liquid medium survived the least well after inoculation of leaf surfaces under all conditions. Cells cultured in solid medium exhibited the highest percent survival and desiccation tolerance in the growth chamber but generally survived less well in the field than did cells harvested from plants. Cells harvested from plants and inoculated onto plants in the field usually exhibited the highest percent survival, started to increase in population earlier, and reached a higher number than did cells cultured in vitro. Differences in field survival were apparently not attributable to differential UV tolerance. The observed effects of phenotypic plasticity on epiphytic survival and colonization should be considered in risk assessment studies, in studies of bacterial epidemiology, and in the use of microbial antagonists for biological pest control.     

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.     

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.     

Survival, Growth, and Localization of Epiphytic Fitness Mutants of Pseudomonas syringae on Leaves

Among 82 epiphytic fitness mutants of a Pseudomonas syringae pv. syringae strain that were characterized in a previous study, 4 mutants were particularly intolerant of the stresses associated with dry leaf surfaces. These four mutants each exhibited distinctive behaviors when inoculated onto and into plant leaves. For example, while none showed measurable growth on dry potato leaf surfaces, they grew to different population sizes in the intercellular spaces of bean leaves and on dry bean leaf surfaces, and one mutant appeared incapable of growth in both environments although it grew well on moist bean leaves. The presence of the parental strain did not influence the survival of the mutants immediately following exposure of leaves to dry, high-light incubation conditions, suggesting that the reduced survival of the mutants did not result from an inability to produce extracellular factors in planta. On moist bean leaves that were colonized by either a mutant or the wild type, the proportion of the total epiphytic population that was located in sites protected from a surface sterilant was smaller for the mutants than for the wild type, indicating that the mutants were reduced in their ability to locate, multiply in, and/or survive in such protected sites. This reduced ability was only one of possibly several factors contributing to the reduced epiphytic fitness of each mutant. Their reduced fitness was not specific to the host plant bean, since they also exhibited reduced fitness on the nonhost plant potato; the functions altered in these strains are thus of interest for their contribution to the general fitness of bacterial epiphytes.     

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.     

Roadmap to new virulence determinants in Pseudomonas syringae: insights from comparative genomics and genome organization

Systematic comparison of the current repertoire of virulence-associated genes for three Pseudomonas syringae strains with complete genome sequences, P. syringae pv. tomato DC3,000, P. syringae pv. phaseolicola 1448A, and P. syringae pv. syringae B728a, is prompted by recent advances in virulence factor identification in P. syringae and other bacteria. Among these are genes linked to epiphytic fitness, plant- and insect-active toxins, secretion pathways, and virulence regulators, all reflected in the recently updated DC3,000 genome annotation. Distribution of virulence genes in relation to P. syringae genome organization was analyzed to distinguish patterns of conservation among genomes and association between genes and mobile genetic elements. Variable regions were identified on the basis of deviation in sequence composition and gaps in syntenic alignment among the three genomes. Mapping gene location relative to the genome structure revealed strong segregation of the HrpL regulon with variable genome regions (VR), divergent distribution patterns for toxin genes depending on association with plant or insect pathogenesis, and patterns of distribution for other virulence genes that highlight potential sources of strain-to-strain differences in host interaction. Distribution of VR among other sequenced bacterial genomes was analyzed and future plans for characterization of this potential reservoir of virulence genes are discussed.     

Pathovar-specific requirement for the Pseudomonas syringae lemA gene in disease lesion formation.

The lemA gene is conserved among strains and pathovars of Pseudomonas syringae. In P. syringae pv. syringae B728a, a causal agent of bacterial brown spot disese of bean, the lemA gene is required for lesion formation on leaves and pods. Using lemA-containing DNA as a probe, we determined that 80 P. syringae pv. syringae strains isolated from bean leaves could be grouped into seven classes based on restriction fragment length polymorphism. Marker exchange mutagenesis showed that the lemA gene was required for lesion formation by representative strains from each restriction fragment length polymorphism class. Hybridization to the lemA locus was detected within six different P. syringae pathovars and within Pseudomonas aeruginosa. Interestingly, a lemA homolog was present and functional within the nonpathogenic strain P. syringae Cit7. We cloned a lemA homolog from a genomic library of P. syringae pv. phaseolicola NPS3121, a causal agent of halo blight of bean, that restored lesion formation to a P. syringae pv. syringae lemA mutant. However, a lemA mutant P. syringae pv. phaseolicola strain retained the ability to produce halo blight disease symptoms on bean plants. Therefore, the lemA gene played an essential role in disease lesion formation by P. syringae pv. syringae isolates, but was not required for pathogenicity of a P. syringae pv. phaseolicola strain.     

Pseudomonas syringae exchangeable effector loci: sequence diversity in representative pathovars and virulence function in P. syringae pv. syringae B728a

Pseudomonas syringae is a plant pathogen whose pathogenicity and host specificity are thought to be determined by Hop/Avr effector proteins injected into plant cells by a type III secretion system. P. syringae pv. syringae B728a, which causes brown spot of bean, is a particularly well-studied strain. The type III secretion system in P. syringae is encoded by hrp (hypersensitive response and pathogenicity) and hrc (hrp conserved) genes, which are clustered in a pathogenicity island with a tripartite structure such that the hrp/hrc genes are flanked by a conserved effector locus and an exchangeable effector locus (EEL). The EELs of P. syringae pv. syringae B728a, P. syringae strain 61, and P. syringae pv. tomato DC3000 differ in size and effector gene composition; the EEL of P. syringae pv. syringae B728a is the largest and most complex. The three putative effector proteins encoded by the P. syringae pv. syringae B728a EEL--HopPsyC, HopPsyE, and HopPsyV--were demonstrated to be secreted in an Hrp-dependent manner in culture. Heterologous expression of hopPsyC, hopPsyE, and hopPsyV in P. syringae pv. tabaci induced the hypersensitive response in tobacco leaves, demonstrating avirulence activity in a nonhost plant. Deletion of the P. syringae pv. syringae B728a EEL strongly reduced virulence in host bean leaves. EELs from nine additional strains representing nine P. syringae pathovars were isolated and sequenced. Homologs of avrPphE (e.g., hopPsyE) and hopPsyA were particularly common. Comparative analyses of these effector genes and hrpK (which flanks the EEL) suggest that the EEL effector genes were acquired by horizontal transfer after the acquisition of the hrp/hrc gene cluster but before the divergence of modern pathovars and that some EELs underwent transpositions yielding effector exchanges or point mutations producing effector pseudogenes after their acquisition.     

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.     

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

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.     

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

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.     

Reduction of olive knot disease by a bacteriocin from Pseudomonas syringae pv. ciccaronei

A bacteriocin produced by Pseudomonas syringae pv. ciccaronei, used at different purification levels and concentrations in culture and in planta, inhibited the multiplication of P. syringae subsp. savastanoi, the causal agent of olive knot disease, and affected the epiphytic survival of the pathogen on the leaves and twigs of treated olive plants. Treatments with bacteriocin from P. syringae pv. ciccaronei inhibited the formation of overgrowths on olive plants caused by P. syringae subsp. savastanoi strains PVBa229 and PVBa304 inoculated on V-shaped slits and on leaf scars at concentrations of 10(5) and 10(8) CFU ml(-1), respectively. In particular, the application of 6,000 arbitrary units (AU) of crude bacteriocin (dialyzed ammonium sulfate precipitate of culture supernatant) ml(-1) at the inoculated V-shaped slits and leaf scars resulted in the formation of knots with weight values reduced by 81 and 51%, respectively, compared to the control, depending on the strains and inoculation method used. Crude bacteriocin (6,000 AU ml(-1)) was also effective in controlling the multiplication of epiphytic populations of the pathogen. In particular, the bacterial populations recovered after 30 days were at least 350 and 20 times lower than the control populations on twigs and on leaves, respectively. These results suggest that bacteriocin from P. syringae pv. ciccaronei can be used effectively to control the survival of the causal agent of olive knot disease and to prevent its multiplication at inoculation sites.     

Type III secretion and effectors shape the survival and growth pattern of Pseudomonas syringae on leaf surfaces

The bacterium Pseudomonas syringae pv syringae B728a (PsyB728a) uses a type III secretion system (T3SS) to inject effector proteins into plant cells, a process that modulates the susceptibility of different plants to infection. Analysis of GREEN FLUORESCENT PROTEIN-expressing PsyB728a after spray inoculation without additives under moderate relative humidity conditions permitted (1) a detailed analysis of this strain's survival and growth pattern on host (Nicotiana benthamiana) and nonhost (tomato [Solanum lycopersicum]) leaf surfaces, (2) an assessment of the role of plant defenses in affecting PsyB728a leaf surface (epiphytic) growth, and (3) the contribution of the T3SS and specific effectors to PsyB728a epiphytic survival and growth. On host leaf surfaces, PsyB728a cells initially persist without growing, and show an increased population only after 48 h, unless plants are pretreated with the defense-inducing chemical benzothiazole. During the persistence period, some PsyB728a cells induce a T3SS reporter, whereas a T3SS-deficient mutant shows reduced survival. By 72 h, rare invasion by PsyB728a to the mesophyll region of host leaves occurs, but endophytic and epiphytic bacterial growths are not correlated. The effectors HopZ3 and HopAA1 delay the onset of epiphytic growth of PsyB728a on N. benthamiana, whereas they promote epiphytic survival/growth on tomato. These effectors localize to distinct sites in plant cells and likely have different mechanisms of action. HopZ3 may enzymatically modify host targets, as it requires residues important for the catalytic activity of other proteins in its family of proteases. Thus, the T3SS, HopAA1, HopZ3, and plant defenses strongly influence epiphytic survival and/or growth of PsyB728a.