Pseudomonas syringae on Plants in Iceland Has Likely Evolved for Several Million Years Outside the Reach of Processes That Mix This Bacterial Complex across Earth's Temperate Zones.

Here we report, for the first time, the occurrence of the bacteria from the species complex Pseudomonas syringae in Iceland. We isolated this bacterium from 35 of the 38 samples of angiosperms, moss, ferns and leaf litter collected across the island from five habitat categories (boreal heath, forest, subalpine and glacial scrub, grazed pasture, lava field). The culturable populations of P. syringae on these plants varied in size across 6 orders of magnitude, were as dense as 107 cfu g-1 and were composed of strains in phylogroups 1, 2, 4, 6, 7, 10 and 13. P. syringae densities were significantly greatest on monocots compared to those on dicots and mosses and were about two orders of magnitude greater in grazed pastures compared to all other habitats. The phylogenetic diversity of 609 strains of P. syringae from Iceland was compared to that of 933 reference strains of P. syringae from crops and environmental reservoirs collected from 27 other countries based on a 343 bp sequence of the citrate synthase (cts) housekeeping gene. Whereas there were examples of identical cts sequences across multiple countries and continents among the reference strains indicating mixing among these countries and continents, the Icelandic strains grouped into monophyletic lineages that were unique compared to all of the reference strains. Based on estimates of the time of divergence of the Icelandic genetic lineages of P. syringae, the geological, botanical and land use history of Iceland, and atmospheric circulation patterns, we propose scenarios whereby it would be feasible for P. syringae to have evolved outside the reach of processes that tend to mix this bacterial complex across the planet elsewhere.

Isolation and identification of Pseudomonas syringae facilitated by a PCR targeting the whole P. syringae group.

We present a reliable PCR-based method to avoid the biases related to identification based on the conventional phenotypes currently used in the identification of Pseudomonas syringae sensu lato, a ubiquitous environmental bacterium including plant pathogens. We identified a DNA target suitable for this purpose by applying a comparative genomic pipeline to Pseudomonas genomes. We designed primers and developed PCR conditions that led to a clean and strong PCR product from 97% of the 185 strains of P. syringae strains tested and gave a clear negative result for the 31 non-P. syringae strains tested. The sensitivity of standard PCR was determined with pure strains to be 10(6) bacteria mL(-1) or 0.4 ng of DNA µL(-1). Sensitivity could be improved with the touchdown method. The new PCR-assisted isolation of P. syringae was efficient when deployed on an environmental sample of river water as compared to the isolation based on phenotypes. This innovation eliminates the need for extensive expertise in isolating P. syringae colonies, was simpler, faster and very reliable. It will facilitate discovery of more diversity of P. syringae and research on emergence, dispersion and evolution to understand the varied functions of this environmental bacterium.

A framework to gauge the epidemic potential of plant pathogens in environmental reservoirs: the example of kiwifruit canker.

New economically important diseases on crops and forest trees emerge recurrently. An understanding of where new pathogenic lines come from and how they evolve is fundamental for the deployment of accurate surveillance methods. We used kiwifruit bacterial canker as a model to assess the importance of potential reservoirs of new pathogenic lineages. The current kiwifruit canker epidemic is at least the fourth outbreak of the disease on kiwifruit caused by Pseudomonas syringae in the mere 50 years in which this crop has been cultivated worldwide, with each outbreak being caused by different genetic lines of the bacterium. Here, we ask whether strains in natural (non-agricultural) environments could cause future epidemics of canker on kiwifruit. To answer this question, we evaluated the pathogenicity, endophytic colonization capacity and competitiveness on kiwifruit of P. syringae strains genetically similar to epidemic strains and originally isolated from aquatic and subalpine habitats. All environmental strains possessing an operon involved in the degradation of aromatic compounds via the catechol pathway grew endophytically and caused symptoms in kiwifruit vascular tissue. Environmental and epidemic strains showed a wide host range, revealing their potential as future pathogens of a variety of hosts. Environmental strains co-existed endophytically with CFBP 7286, an epidemic strain, and shared about 20 virulence genes, but were missing six virulence genes found in all epidemic strains. By identifying the specific gene content in genetic backgrounds similar to known epidemic strains, we developed criteria to assess the epidemic potential and to survey for such strains as a means of forecasting and managing disease emergence.

A user's guide to a data base of the diversity of Pseudomonas syringae and its application to classifying strains in this phylogenetic complex.

The Pseudomonas syringae complex is composed of numerous genetic lineages of strains from both agricultural and environmental habitats including habitats closely linked to the water cycle. The new insights from the discovery of this bacterial species in habitats outside of agricultural contexts per se have led to the revelation of a wide diversity of strains in this complex beyond what was known from agricultural contexts. Here, through Multi Locus Sequence Typing (MLST) of 216 strains, we identified 23 clades within 13 phylogroups among which the seven previously described P. syringae phylogroups were included. The phylogeny of the core genome of 29 strains representing nine phylogroups was similar to the phylogeny obtained with MLST thereby confirming the robustness of MLST-phylogroups. We show that phenotypic traits rarely provide a satisfactory means for classification of strains even if some combinations are highly probable in some phylogroups. We demonstrate that the citrate synthase (cts) housekeeping gene can accurately predict the phylogenetic affiliation for more than 97% of strains tested. We propose a list of cts sequences to be used as a simple tool for quickly and precisely classifying new strains. Finally, our analysis leads to predictions about the diversity of P. syringae that is yet to be discovered. We present here an expandable framework mainly based on cts genetic analysis into which more diversity can be integrated.

The Pseudomonas viridiflava phylogroups in the P. syringae species complex are characterized by genetic variability and phenotypic plasticity of pathogenicity-related traits.

As a species complex, Pseudomonas syringae exists in both agriculture and natural aquatic habitats. P.viridiflava, a member of this complex, has been reported to be phenotypically largely homogenous. We characterized strains from different habitats, selected based on their genetic similarity to previously described P.viridiflava strains. We revealed two distinct phylogroups and two different kinds of variability in phenotypic traits and genomic content. The strains exhibited phase variation in phenotypes including pathogenicity and soft rot on potato. We showed that the presence of two configurations of the Type III Secretion System [single (S-PAI) and tripartite (T-PAI) pathogenicity islands] are not correlated with pathogenicity or with the capacity to induce soft rot in contrast to previous reports. The presence/absence of the avrE effector gene was the only trait we found to be correlated with pathogenicity of P.viridiflava. Other Type III secretion effector genes were not correlated with pathogenicity. A genomic region resembling an exchangeable effector locus (EEL) was found in S-PAI strains, and a probable recombination between the two PAIs is described. The ensemble of the variability observed in these phylogroups of P.syringae likely contributes to their adaptability to alternating opportunities for pathogenicity or saprophytic survival.

Draft Genome Sequences of a Phylogenetically Diverse Suite of Pseudomonas syringae Strains from Multiple Source Populations.

Here, we report the draft genome sequences for 7 phylogenetically diverse isolates of Pseudomonas syringae, obtained from numerous environmental sources and geographically proximate crop species. Overall, these sequences provide a wealth of information about the differences (or lack thereof) between isolates from disease outbreaks and those from other sources.

Pseudomonas syringae naturally lacking the canonical type III secretion system are ubiquitous in nonagricultural habitats, are phylogenetically diverse and can be pathogenic.

The type III secretion system (T3SS) is an important virulence factor of pathogenic bacteria, but the natural occurrence of variants of bacterial plant pathogens with deficiencies in their T3SS raises questions about the significance of the T3SS for fitness. Previous work on T3SS-deficient plant pathogenic bacteria has focused on strains from plants or plant debris. Here we have characterized T3SS-deficient strains of Pseudomonas syringae from plant and nonplant substrates in pristine nonagricultural contexts, many of which represent recently described clades not yet found associated with crop plants. Strains incapable of inducing a hypersensitive reaction (HR(-)) in tobacco were detected in 65% of 126 samples from headwaters of rivers (mountain creeks and lakes), snowpack, epilithic biofilms, wild plants and leaf litter and constituted 2 to 100% of the P. syringae population associated with each sample. All HR(-) strains lacked at least one gene in the canonical hrp/hrc locus or the associated conserved effector locus, but most lacked all six of the genes tested (hrcC, hrpL, hrpK1, avrE1 and hrpW1) and represented several disparate phylogenetic clades. Although most HR(-) strains were incapable of causing symptoms on cantaloupe seedlings as expected, strains in the recently described TA-002 clade caused severe symptoms in spite of the absence of any of the six conserved genes of the canonical T3SS according to PCR and Southern blot assays. The phylogenetic context of the T3SS variants we observed provides insight into the evolutionary history of P. syringae as a pathogen and as an environmental saprophyte.

Emigration of the plant pathogen Pseudomonas syringae from leaf litter contributes to its population dynamics in alpine snowpack.

The recently discovered ubiquity of the plant pathogen Pseudomonas syringae in headwaters and alpine ecosystems worldwide elicits new questions about the ecology of this bacterium and subsequent consequences for disease epidemiology. Because of the major contribution of snow to river run-off during crop growth, we evaluated the population dynamics of P.syringae in snowpack and the underlying leaf litter during two years in the Southern French Alps. High population densities of P.syringae were found on alpine grasses, and leaf litter was identified as the main source of populations of P.syringae in snowpack, contributing more than the populations arriving with the snowfall. The insulating properties of snow foster survival of P.syringae throughout the winter in the 10 cm layer of snow closest to the soil. Litter and snowpack harboured populations of P.syringae that were very diverse in terms of phenotypes and genotypes. Neither substrate nor sampling site had a marked effect on the structure of P.syringae populations, and snow and litter had genotypes in common with other non-agricultural habitats and with crops. These results contribute to the mounting evidence that a highly diverse P.syringae metapopulation is disseminated throughout drainage basins between cultivated and non-cultivated zones.

Broad Diversity of Ralstonia solanacearum Strains in Cameroon.

In 2005, an extensive survey of bacterial wilt in Cameroon collected 110 strains of Ralstonia solanacearum from wilting tomato, potato, pepper, huckleberry (Solanum scabrum), sesame, and amaranth. The genetic diversity and phylogeny of selected strains from Cameroon were assessed by multiplex-polymerase chain reaction (PCR), race 3/biovar 2-specific PCR, and sequence analyses of the mutS and egl genes. These data were compared with those from 33 reference strains covering the known diversity within the R. solanacearum species complex. Strains isolated in Cameroon clustered into three of the four known phylotypes: I (Asian), II (American), and III (African). Lowland tomato strains belonged to phylotype I and were quite homogeneous. The strains belonging to phylotype II were genetically diverse, and partitioned into subclusters IIA and IIB (sequevar 1, race 3/biovar 2). Cameroon strains in the African phylotype III were distinct from reference strains from Zimbabwe or the Indian Ocean, highlighting the genetic diversity present within this phylotype. Strains from potatoes growing in the highlands of West Cameroon fell into both phylotypes II (race 3/biovar 2) and III. These phylotype II and III highland strains attacked both potato and tomato and could therefore pose an economic threat to potato and tomato crops throughout Central Africa. This is the first comprehensive report on the genetic diversity of R. solanacearum strains in Cameroon.

The life history of the plant pathogen Pseudomonas syringae is linked to the water cycle.

Pseudomonas syringae is a plant pathogen well known for its capacity to grow epiphytically on diverse plants and for its ice-nucleation activity. The ensemble of its known biology and ecology led us to postulate that this bacterium is also present in non-agricultural habitats, particularly those associated with water. Here, we report the abundance of P. syringae in rain, snow, alpine streams and lakes and in wild plants, in addition to the previously reported abundance in epilithic biofilms. Each of these substrates harbored strains that corresponded to P. syringae in terms of biochemical traits, pathogenicity and pathogenicity-related factors and that were ice-nucleation active. Phylogenetic comparisons of sequences of four housekeeping genes of the non-agricultural strains with strains of P. syringae from disease epidemics confirmed their identity as P. syringae. Moreover, strains belonging to the same clonal lineage were isolated from snow, irrigation water and a diseased crop plant. Our data suggest that the different substrates harboring P. syringae modify the structure of the associated populations. Here, we propose a comprehensive life cycle for P. syringae--in agricultural and non-agricultural habitats--driven by the environmental cycle of water. This cycle opens the opportunity to evaluate the importance of non-agricultural habitats in the evolution of a plant pathogen and the emergence of virulence. The ice-nucleation activity of all strains from snow, unlike from other substrates, strongly suggests that P. syringae plays an active role in the water cycle as an ice nucleus in clouds.

Ralstonia solanacearum strains from Martinique (French West Indies) exhibiting a new pathogenic potential.

We investigated a destructive pathogenic variant of the plant pathogen Ralstonia solanacearum that was consistently isolated in Martinique (French West Indies). Since the 1960s, bacterial wilt of solanaceous crops in Martinique has been caused primarily by strains of R. solanacearum that belong to either phylotype I or phylotype II. Since 1999, anthurium shade houses have been dramatically affected by uncharacterized phylotype II strains that also affected a wide range of species, such as Heliconia caribea, cucurbitaceous crops, and weeds. From 1989 to 2003, a total of 224 R. solanacearum isolates were collected and compared to 6 strains isolated in Martinique in the 1980s. The genetic diversity and phylogenetic position of selected strains from Martinique were assessed (multiplex PCRs, mutS and egl DNA sequence analysis) and compared to the genetic diversity and phylogenetic position of 32 reference strains covering the known diversity within the R. solanacearum species complex. Twenty-four representative isolates were tested for pathogenicity to Musa species (banana) and tomato, eggplant, and sweet pepper. Based upon both PCR and sequence analysis, 119 Martinique isolates from anthurium, members of the family Cucurbitaceae, Heliconia, and tomato, were determined to belong to a group termed phylotype II/sequevar 4 (II/4). While these strains cluster with the Moko disease-causing strains, they were not pathogenic to banana (NPB). The strains belonging to phylotype II/4NPB were highly pathogenic to tomato, eggplant, and pepper, were able to wilt the resistant tomato variety Hawaii7996, and may latently infect cooking banana. Phylotype II/4NPB constitutes a new pathogenic variant of R. solanacearum that has recently appeared in Martinique and may be latently prevalent throughout Caribbean and Central/South America.