Recently, genome sequencing of many isolates of genetically monomorphic bacterial human pathogens has given new insights into pathogen microevolution and phylogeography. Here, we report a genome-based micro-evolutionary study of a bacterial plant pathogen, Pseudomonas syringae pv. tomato. Only 267 mutations were identified between five sequenced isolates in 3,543,009 nt of analyzed genome sequence, which suggests a recent evolutionary origin of this pathogen. Further analysis with genome-derived markers of 89 world-wide isolates showed that several genotypes exist in North America and in Europe indicating frequent pathogen movement between these world regions. Genome-derived markers and molecular analyses of key pathogen loci important for virulence and motility both suggest ongoing adaptation to the tomato host. A mutational hotspot was found in the type III-secreted effector gene hopM1. These mutations abolish the cell death triggering activity of the full-length protein indicating strong selection for loss of function of this effector, which was previously considered a virulence factor. Two non-synonymous mutations in the flagellin-encoding gene fliC allowed identifying a new microbe associated molecular pattern (MAMP) in a region distinct from the known MAMP flg22. Interestingly, the ancestral allele of this MAMP induces a stronger tomato immune response than the derived alleles. The ancestral allele has largely disappeared from today's Pto populations suggesting that flagellin-triggered immunity limits pathogen fitness even in highly virulent pathogens. An additional non-synonymous mutation was identified in flg22 in South American isolates. Therefore, MAMPs are more variable than expected differing even between otherwise almost identical isolates of the same pathogen strain. Our knowledge of the recent evolution of bacterial human pathogens has increased dramatically over the last five years. By comparison, relatively little is known about recent evolution of bacterial plant pathogens. Here, we analyze a large collection of isolates of the economically important plant pathogen Pseudomonas syringae pv. tomato with markers derived from the comparison of five genomes of this pathogen. We find that this pathogen likely evolved on a relatively recent time scale and continues to adapt to tomato by minimizing its recognition by the tomato immune system. We find that an allele of the flagellin subunit fliC that appeared in the pathogen population for the first time in the 1980s, and which is the most common allele of this gene in North America and Europe today, triggers a weaker tomato immune response than the fliC allele found in the 1960s and 1970s. These results not only impact our understanding of pathogen – plant interactions and pathogen evolution but also have important ramifications for disease prevention. Given the speed with which new pathogen strains spread and replace existing strains, limiting the movement of specific strains between geographic regions is critically important, even for pathogens known to have worldwide distribution.

Background: Bacterial spot of tomato and pepper is caused by four Xanthomonas species and is a major plant disease in warm humid climates. The four species are distinct from each other based on physiological and molecular characteristics. The genome sequence of strain 85-10, a member of one of the species, Xanthomonas euvesicatoria (Xcv) has been previously reported. To determine the relationship of the four species at the genome level and to investigate the molecular basis of their virulence and differing host ranges, draft genomic sequences of members of the other three species were determined and compared to strain 85-10. Results: We sequenced the genomes of X. vesicatoria (Xv) strain 1111 (ATCC 35937), X. perforans (Xp) strain 91-118 and X. gardneri (Xg) strain 101 (ATCC 19865). The genomes were compared with each other and with the previously sequenced Xcv strain 85-10. In addition, the molecular features were predicted that may be required for pathogenicity including the type III secretion apparatus, type III effectors, other secretion systems, quorum sensing systems, adhesins, extracellular polysaccharide, and lipopolysaccharide determinants. Several novel type III effectors from Xg strain 101 and Xv strain 1111 genomes were computationally identified and their translocation was validated using a reporter gene assay. A homolog to Ax21, the elicitor of XA21-mediated resistance in rice, and a functional Ax21 sulfation system were identified in Xcv. Genes encoding proteins with functions mediated by type II and type IV secretion systems have also been compared, including enzymes involved in cell wall deconstruction, as contributors to pathogenicity. Conclusions: Comparative genomic analyses revealed considerable diversity among bacterial spot pathogens, providing new insights into differences and similarities that may explain the diverse nature of these strains. Genes specific to pepper pathogens, such as the O-antigen of the lipopolysaccharide cluster, and genes unique to individual strains, such as novel type III effectors and bacteriocin genes, have been identified providing new clues for our understanding of pathogen virulence, aggressiveness, and host preference. These analyses will aid in efforts towards breeding for broad and durable resistance in economically important tomato and pepper cultivars.

Summary: The bacterial flagellin (FliC) epitopes flg22 and flgII‐28 are microbe‐associated molecular patterns (MAMPs). Although flg22 is recognized by many plant species via the pattern recognition receptor FLS2, neither the flgII‐28 receptor nor the extent of flgII‐28 recognition by different plant families is known. Here, we tested the significance of flgII‐28 as a MAMP and the importance of allelic diversity in flg22 and flgII‐28 in plant–pathogen interactions using purified peptides and a Pseudomonas syringae ∆fliC mutant complemented with different fliC alleles. The plant genotype and allelic diversity in flg22 and flgII‐28 were found to significantly affect the plant immune response, but not bacterial motility. The recognition of flgII‐28 is restricted to a number of solanaceous species. Although the flgII‐28 peptide does not trigger any immune response in Arabidopsis, mutations in both flg22 and flgII‐28 have FLS2‐dependent effects on virulence. However, the expression of a tomato allele of FLS2 does not confer to Nicotiana benthamiana the ability to detect flgII‐28, and tomato plants silenced for FLS2 are not altered in flgII‐28 recognition. Therefore, MAMP diversification is an effective pathogen virulence strategy, and flgII‐28 appears to be perceived by an as yet unidentified receptor in the Solanaceae, although it has an FLS2‐dependent virulence effect in Arabidopsis.

Intercontinental spread of emerging plant diseases is one of the most serious threats to world agriculture. One emerging disease is bacterial canker of kiwi fruit (Actinidia deliciosa and A. chinensis) caused by Pseudomonas syringae pv. actinidiae (PSA). The disease first occurred in China and Japan in the 1980s and in Korea and Italy in the 1990s. A more severe form of the disease broke out in Italy in 2008 and in additional countries in 2010 and 2011 threatening the viability of the global kiwi fruit industry. To start investigating the source and routes of international transmission of PSA, genomes of strains from China (the country of origin of the genus Actinidia), Japan, Korea, Italy and Portugal have been sequenced. Strains from China, Italy, and Portugal have been found to belong to the same clonal lineage with only 6 single nucleotide polymorphisms (SNPs) in 3,453,192 bp and one genomic island distinguishing the Chinese strains from the European strains. Not more than two SNPs distinguish each of the Italian and Portuguese strains from each other. The Japanese and Korean strains belong to a separate genetic lineage as previously reported. Analysis of additional European isolates and of New Zealand isolates exploiting genome-derived markers showed that these strains belong to the same lineage as the Italian and Chinese strains. Interestingly, the analyzed New Zealand strains are identical to European strains at the tested SNP loci but test positive for the genomic island present in the sequenced Chinese strains and negative for the genomic island present in the European strains. Results are interpreted in regard to the possible direction of movement of the pathogen between countries and suggest a possible Chinese origin of the European and New Zealand outbreaks.

A growing body of circumstantial evidence suggests that ice nucleation active (Ice⁺) bacteria contribute to the initiation of precipitation by heterologous freezing of super-cooled water in clouds. However, little is known about the concentration of Ice⁺ bacteria in precipitation, their genetic and phenotypic diversity, and their relationship to air mass trajectories and precipitation chemistry. In this study, 23 precipitation events were collected over 15 months in Virginia, USA. Air mass trajectories and water chemistry were determined and 33 134 isolates were screened for ice nucleation activity (INA) at −8 °C. Of 1144 isolates that tested positive during initial screening, 593 had confirmed INA at −8 °C in repeated tests. Concentrations of Ice⁺ strains in precipitation were found to range from 0 to 13 219 colony forming units per liter, with a mean of 384±147. Most Ice⁺ bacteria were identified as members of known and unknown Ice⁺ species in the Pseudomonadaceae, Enterobacteriaceae and Xanthomonadaceae families, which nucleate ice employing the well-characterized membrane-bound INA protein. Two Ice⁺ strains, however, were identified as Lysinibacillus, a Gram-positive genus not previously known to include Ice⁺ bacteria. INA of the Lysinibacillus strains is due to a nanometer-sized molecule that is heat resistant, lysozyme and proteinase resistant, and secreted. Ice⁺ bacteria and the INA mechanisms they employ are thus more diverse than expected. We discuss to what extent the concentration of culturable Ice⁺ bacteria in precipitation and the identification of a new heat-resistant biological INA mechanism support a role for Ice⁺ bacteria in the initiation of precipitation.

Background: Citrus canker is a disease that has severe economic impact on the citrus industry worldwide. There are three types of canker, called A, B, and C. The three types have different phenotypes and affect different citrus species. The causative agent for type A is Xanthomonas citri subsp. citri, whose genome sequence was made available in 2002. Xanthomonas fuscans subsp. aurantifolii strain B causes canker B and Xanthomonas fuscans subsp. aurantifolii strain C causes canker C. Results: We have sequenced the genomes of strains B and C to draft status. We have compared their genomic content to X. citri subsp. citri and to other Xanthomonas genomes, with special emphasis on type III secreted effector repertoires. In addition to pthA, already known to be present in all three citrus canker strains, two additional effector genes, xopE3 and xopAI, are also present in all three strains and are both located on the same putative genomic island. These two effector genes, along with one other effector-like gene in the same region, are thus good candidates for being pathogenicity factors on citrus. Numerous gene content differences also exist between the three cankers strains, which can be correlated with their different virulence and host range. Particular attention was placed on the analysis of genes involved in biofilm formation and quorum sensing, type IV secretion, flagellum synthesis and motility, lipopolysacharide synthesis, and on the gene xacPNP, which codes for a natriuretic protein. Conclusion: We have uncovered numerous commonalities and differences in gene content between the genomes of the pathogenic agents causing citrus canker A, B, and C and other Xanthomonas genomes. Molecular genetics can now be employed to determine the role of these genes in plant-microbe interactions. The gained knowledge will be instrumental for improving citrus canker control.