Naïve Bayes Classifiers and accompanying dataset for Pseudomonas syringae isolate characterization.

The Pseudomonas syringae species complex (PSSC) is a diverse group of plant pathogens with a collective host range encompassing almost every food crop grown today. As a threat to global food security, rapid detection and characterization of epidemic and emerging pathogenic lineages is essential. However, phylogenetic identification is often complicated by an unclarified and ever-changing taxonomy, making practical use of available databases and the proper training of classifiers difficult. As such, while amplicon sequencing is a common method for routine identification of PSSC isolates, there is no efficient method for accurate classification based on this data. Here we present a suite of five Naïve bayes classifiers for PCR primer sets widely used for PSSC identification, trained on in-silico amplicon data from 2,161 published PSSC genomes using the life identification number (LIN) hierarchical clustering algorithm in place of traditional Linnaean taxonomy. Additionally, we include a dataset for translating classification results back into traditional taxonomic nomenclature (i.e. species, phylogroup, pathovar), and for predicting virulence factor repertoires.

Variation at the common polysaccharide antigen locus drives lipopolysaccharide diversity within the Pseudomonas syringae species complex.

The common polysaccharide antigen (CPA) of the lipopolysaccharide (LPS) from Pseudomonas syringae is highly variable, but the genetic basis for this is poorly understood. We have characterized the CPA locus from P. syringae pv. actinidiae (Psa). This locus has genes for l- and d-rhamnose biosynthesis and an operon coding for ABC transporter subunits, a bifunctional glycosyltransferase and an o-methyltransferase. This operon is predicted to have a role in the transport, elongation and termination of the CPA oligosaccharide and is referred to as the TET operon. Two alleles of the TET operon were present in different biovars (BV) of Psa and lineages of the closely related pathovar P. syringae pv. actinidifoliorum. This allelic variation was reflected in the electrophoretic properties of purified LPS from the different isolates. Gene knockout of the TET operon allele from BV1 and replacement with that from BV3, demonstrated the link between the genetic locus and the biochemical properties of the LPS molecules in Psa. Sequence analysis of the TET operon from a range of P. syringae and P. viridiflava isolates displayed a phylogenetic history incongruent with core gene phylogeny but correlates with previously reported tailocin sensitivity, suggesting a functional relationship between LPS structure and tailocin susceptibility.

Comparative transcriptomic analysis of global gene expression mediated by (p) ppGpp reveals common regulatory networks in Pseudomonas syringae.

BACKGROUND: Pseudomonas syringae is an important plant pathogen, which could adapt many different environmental conditions. Under the nutrient-limited and other stress conditions, P. syringae produces nucleotide signal molecules, i.e., guanosine tetra/pentaphosphate ((p)ppGpp), to globally regulate gene expression. Previous studies showed that (p) ppGpp played an important role in regulating virulence factors in P. syringae pv. tomato DC3000 (PstDC3000) and P. syringae pv. syringae B728a (PssB728a). Here we present a comparative transcriptomic analysis to uncover the overall effects of (p)ppGpp-mediated stringent response in P. syringae. RESULTS: In this study, we investigated global gene expression profiles of PstDC3000 and PssB728a and their corresponding (p)ppGpp0 mutants in hrp-inducing minimal medium (HMM) using RNA-seq. A total of 1886 and 1562 differentially expressed genes (DEGs) were uncovered between the (p)ppGpp0 mutants and the wild-type in PstDC3000 and PssB728a, respectively. Comparative transcriptomics identified 1613 common DEGs, as well as 444 and 293 unique DEGs in PstDC3000 and PssB728a, respectively. Functional cluster analysis revealed that (p) ppGpp positively regulated a variety of virulence-associated genes, including type III secretion system (T3SS), type VI secretion system (T6SS), cell motility, cell division, and alginate biosynthesis, while negatively regulated multiple basic physiological processes, including DNA replication, RNA processes, nucleotide biosynthesis, fatty acid metabolism, ribosome protein biosynthesis, and amino acid metabolism in both PstDC3000 and PssB728a. Furthermore, (p) ppGpp had divergent effects on other processes in PstDC3000 and PssB728a, including phytotoxin, nitrogen regulation and general secretion pathway (GSP). CONCLUSION: In this study, comparative transcriptomic analysis reveals common regulatory networks in both PstDC3000 and PssB728a mediated by (p) ppGpp in HMM. In both P. syringae systems, (p) ppGpp re-allocate cellular resources by suppressing multiple basic physiological activities and enhancing virulence gene expression, suggesting a balance between growth, survival and virulence. Our research is important in that due to similar global gene expression mediated by (p) ppGpp in both PstDC3000 and PssB728a, it is reasonable to propose that (p) ppGpp could be used as a target to develop novel control measures to fight against important plant bacterial diseases.

Genomic analyses of Pseudomonas syringae pv. actinidiae isolated in Korea suggest the transfer of the bacterial pathogen via kiwifruit pollen.

Introduction. The bacterial pathogen, Pseudomonas syringae pv. actinidiae (Psa), has emerged as a major threat to kiwifruit cultivation throughout the world. One pandemic strain (from the Psa3 group) has occurred in various geographical regions. It is important to understand how this pathogen is being transmitted.Aim. Although Psa has been found in Korea since 1992, the isolates were until recently of a distinct type (Psa2). Recently, the more virulent Psa3 type has been detected. The purpose of this study was to describe the variety of Psa3 now found in Korea.Methodology. Strains were isolated from kiwifruit plants in Korea and from pollen imported into Korea from New Zealand. The genomes of 10 isolates were sequenced using the Illumina platform and compared to the completely assembled genomes of pandemic Psa3 strains from New Zealand and China. Comparisons were also made with pandemic strains from Chile and non-pandemic Psa3 isolates from China.Results. Six of the 10 Psa3 isolates from Korea show a clear relationship with New Zealand isolates. Two isolates show a distinct relationship to isolates from Chile; one further isolate has a sequence that is highly similar to that of M228, a strain previously isolated in China; and the last isolate belongs to the Psa3 group, but is not a member of the pandemic lineage.Conclusion. This analysis establishes that there have been multiple routes of transmission of the Psa3 pandemic strain into Korea. One route has involved the importation of pollen from New Zealand. A second route probably involves importation from Chile.

The Ptr1 Locus of Solanum lycopersicoides Confers Resistance to Race 1 Strains of Pseudomonas syringae pv. tomato and to Ralstonia pseudosolanacearum by Recognizing the Type III Effectors AvrRpt2 and RipBN.

Race 1 strains of Pseudomonas syringae pv. tomato, which cause bacterial speck disease of tomato, are becoming increasingly common and no simply inherited genetic resistance to such strains is known. We discovered that a locus in Solanum lycopersicoides, termed Pseudomonas tomato race 1 (Ptr1), confers resistance to race 1 P. syringae pv. tomato strains by detecting the activity of type III effector AvrRpt2. In Arabidopsis, AvrRpt2 degrades the RIN4 protein, thereby activating RPS2-mediated immunity. Using site-directed mutagenesis of AvrRpt2, we found that, like RPS2, activation of Ptr1 requires AvrRpt2 proteolytic activity. Ptr1 also detected the activity of AvrRpt2 homologs from diverse bacteria, including one in Ralstonia pseudosolanacearum. The genome sequence of S. lycopersicoides revealed no RPS2 homolog in the Ptr1 region. Ptr1 could play an important role in controlling bacterial speck disease and its future cloning may shed light on an example of convergent evolution for recognition of a widespread type III effector.

Some strains that have converged to infect Prunus spp. trees are members of distinct Pseudomonas syringae genomospecies and ecotypes as revealed by in silico genomic comparison.

A complementary taxonomic and population genetic study was performed to delineate genetically and ecologically distinct species within the Pseudomonas syringae complex by assessing 16 strains including pathovar strains that have converged to infect Prunus spp. trees, and two outgroups. Both average nucleotide identity and genome-to-genome distance comparison methods revealed the occurrence of distinct genomospecies, namely 1, 2, 3 and 8 (sensu Gardan et al.), with the latter two being closely related. Strains classified as P. s. pv. morsprunorum clustered into two distinct genomospecies, namely 2 and 8. Both the AdaptML and hierarchical Bayesian analysis of population structure methods highlighted the presence of three ecotypes, and the taxonomically related genomospecies 3 and 8 strains were members of the same ecotype. The distribution of pathogenic and virulence-associated genetic traits among Pseudomonas strains did not reveal any distinct type III secretion system effector or phytotoxin distribution pattern that characterized single genomospecies and strains that infect Prunus spp. The complete WHOP (Woody HOst and Pseudomonas spp.) genomic region and the entire ß-ketoadipate gene cluster, including the catBCA operon, were found only in the members of genomospecies 2 and in the two P. s. pv. morsprunorum strains of genomospecies 8. A reduced gene flow between the three ecotypes suggested that point mutations played a larger role during the evolution of the strains than recombination. Our data support the idea that Prunus trees can be infected by different strains of distinct Pseudomonas genomospecies/ecotypes through diverse mechanisms of host colonization and infection. Such strains may represent particular lineages that emerged from environments other than that of the infected plant upon acquiring genetic traits that gave them the ability to cause plant diseases. The complementary assessment of bacterial strains using both taxonomic approaches and methods that reveal ecologically homogeneous populations has proven useful in confirming the cohesion of bacterial clusters.

Localized recombination drives diversification of killing spectra for phage-derived syringacins.

To better understand the potential for antagonistic interactions between members of the same bacterial species, we have surveyed bacteriocin killing activity across a diverse suite of strains of the phytopathogen Pseudomonas syringae. Our data demonstrate that killing activity from phage-derived bacteriocins of P. syringae (R-type syringacins) is widespread. Despite a high overall diversity of bacteriocin activity, strains can broadly be classified into five main killing types and two main sensitivity types. Furthermore, we show that killing activity switches frequently between strains and that switches correlate with localized recombination of two genes that together encode the proteins that specify bacteriocin targeting. Lastly, we demonstrate that phage-derived bacteriocin killing activity can be swapped between strains simply through expression of these two genes in trans. Overall, our study characterizes extensive diversity of killing activity for phage-derived bacteriocins of P. syringae across strains and highlights the power of localized recombination to alter phenotypes that mediate strain interactions during evolution of natural populations and communities.

Pseudomonas syringae: enterprising epiphyte and stealthy parasite.

Pseudomonas syringae is best known as a plant pathogenic bacterium that causes diseases in a multitude of hosts, and it has been used as a model organism to understand the biology of plant disease. Pathogenic and non-pathogenic isolates of P. syringae are also commonly found living as epiphytes and in the wider environment, including water sources such as rivers and precipitation. Ice-nucleating strains of P. syringae are associated with frost damage to crops. The genomes of numerous strains of P. syringae have been sequenced and molecular genetic studies have elucidated many aspects of this pathogen's interaction with its host plants.

Characterization of Pseudomonas syringae pv. actinidiae biovar 3 on kiwifruit in north-west Portugal.

AIMS: Bacterial kiwifruit canker disease, caused by Pseudomonas syringae pv. actinidiae (Psa) was detected in north-west Portugal in 2010, and has since caused significant losses. The objectives of this work were to characterize the Portuguese population(s) of Psa and to define the actual prevalence of Psa biovars in the most productive kiwifruit region in Portugal. METHODS AND RESULTS: Isolates obtained from Actinidia deliciosa orchards were characterized by morphological, biochemical, physiological, fatty acids and molecular tests (PCR, BOX-PCR, duplex-PCR, multiplex-PCR and RFLP), phaseolotoxin, housekeeping and effector genes and pathogenicity. Results established that only Psa biovar 3 is present in the north-west of Portugal, despite phenotypic and genetic variability among the isolates. CONCLUSIONS: This work provides new information on P. syringae pv. actinidiae (Psa) genetic profile in Portugal, indicating for the first time, that two genetically different subpopulations of Psa biovar 3 are present. SIGNIFICANCE AND IMPACT OF THE STUDY: A new subpopulation of Psa biovar 3 was found for the first time in Portugal, contributing to increase knowledge about this population worldwide and to support further understanding of the impact of Psa.

Comparative genomics of Pseudomonas syringae reveals convergent gene gain and loss associated with specialization onto cherry (Prunus avium).

Genome-wide analyses of the effector- and toxin-encoding genes were used to examine the phylogenetics and evolution of pathogenicity amongst diverse strains of Pseudomonas syringae causing bacterial canker of cherry (Prunus avium), including pathovars P. syringae pv morsprunorum (Psm) races 1 and 2, P. syringae pv syringae (Pss) and P. syringae pv avii. Phylogenetic analyses revealed Psm races and P. syringae pv avii clades were distinct and were each monophyletic, whereas cherry-pathogenic strains of Pss were interspersed amongst strains from other host species. A maximum likelihood approach was used to predict effectors associated with pathogenicity on cherry. Pss possesses a smaller repertoire of type III effectors but has more toxin biosynthesis clusters than Psm and P. syringae pv avii. Evolution of cherry pathogenicity was correlated with gain of genes such as hopAR1 and hopBB1 through putative phage transfer and horizontal transfer respectively. By contrast, loss of the avrPto/hopAB redundant effector group was observed in cherry-pathogenic clades. Ectopic expression of hopAB and hopC1 triggered the hypersensitive reaction in cherry leaves, confirming computational predictions. Cherry canker provides a fascinating example of convergent evolution of pathogenicity that is explained by the mix of effector and toxin repertoires acting on a common host.

Inactivation of bacterial quorum sensing signals N-acyl homoserine lactones is widespread in yeasts.

The inactivation of quorum sensing signals, a phenomenon known as quorum quenching, has been described in diverse microorganisms, though it remains almost unexplored in yeasts. Beyond the well-known properties of these microorganisms for the industry or as eukaryotic models, the role of yeasts in soil or in the inner tissues of a plant is largely unknown. In this report, the wider survey of quorum quenching activities in yeasts isolated from Antarctic soil and the inner tissues of sugarcane, a tropical crop, is presented. Results show that, independently of their niche, quorum quenching activities are broadly present in unicellular fungi. Although yeasts showing a broad range of quorum quenching activity are present in the two niches, at the same time specific AHL inactivation profiles can also be found. Furthermore, yeasts from both sampling sites show quorum quenching activities compatible with lactonase-like and acylase-like inactivations of AHLs. Interestingly, the characterization of Rhodotorula mucilaginosa 7Apo1 showed that the presence of a particular AHL does not interfere with the quenching of a second molecule. Evidence suggests that yeasts could play a role in the modulation of the quorum sensing activity of bacteria. The relationship among phylogeny, sampling sites and yeast quorum quenching activities of the isolates is analyzed.

Draft Whole Genome Sequence Analyses on Pseudomonas syringae pv. actinidiae Hypersensitive Response Negative Strains Detected from Kiwifruit Bleeding Sap Samples.

Kiwifruit bleeding sap samples, collected in Italian and Chilean orchards from symptomatic and asymptomatic plants, were evaluated for the presence of Pseudomonas syringae pv. actinidiae, the causal agent of bacterial canker. The saps were sampled during the spring in both hemispheres, before the bud sprouting, during the optimal time window for the collection of an adequate volume of sample for the early detection of the pathogen, preliminarily by molecular assays, and then through its direct isolation and identification. The results of molecular analyses showed more effectiveness in the P. syringae pv. actinidiae detection when compared with those of microbiological analyses through the pathogen isolation on the nutritive and semiselective media selected. The bleeding sap analyses allowed the isolation and identification of two hypersensitive response (HR) negative and hypovirulent P. syringae pv. actinidiae strains from different regions in Italy. Moreover, multilocus sequence analysis (MLSA) and whole genome sequence (WGS) were carried out on selected Italian and Chilean P. syringae pv. actinidiae virulent strains to verify the presence of genetic variability compared with the HR negative strains and to compare the variability of selected gene clusters between strains isolated in both countries. All the strains showed the lack of argK and coronatine gene clusters as reported for the biovar 3 P. syringae pv. actinidiae strains. Despite the biologic differences obtained in the tobacco bioassays and in pathogenicity assays, the MLSA and WGS analyses did not show significant differences between the WGS of the HR negative and HR positive strains; the difference, on the other hand, between PAC_ICE sequences of Italian and Chilean P. syringae pv. actinidiae strains was confirmed. The inability of the hypovirulent strains IPV-BO 8893 and IPV-BO 9286 to provoke HR in tobacco and the low virulence shown in this host could not be associated with mutations or recombinations in T3SS island.

Pseudomonas caspiana sp. nov., a citrus pathogen in the Pseudomonas syringae phylogenetic group.

In a screening by multilocus sequence analysis of Pseudomonas strains isolated from diverse origins, 4 phylogenetically closely related strains (FBF58, FBF102T, FBF103, and FBF122) formed a well-defined cluster in the Pseudomonas syringae phylogenetic group. The strains were isolated from citrus orchards in northern Iran with disease symptoms in the leaves and stems and its pathogenicity against citrus plants was demonstrated. The whole genome of the type strain of the proposed new species (FBF102T=CECT 9164T=CCUG 69273T) was sequenced and characterized. Comparative genomics with the 14 known Pseudomonas species type strains of the P. syringae phylogenetic group demonstrated that this strain belonged to a new genomic species, different from the species described thus far. Genome analysis detected genes predicted to be involved in pathogenesis, such as an atypical type 3 secretion system and two type 6 secretion systems, together with effectors and virulence factors. A polyphasic taxonomic characterization demonstrated that the 4 plant pathogenic strains represented a new species, for which the name Pseudomonas caspiana sp. nov. is proposed.

Evidence for a Novel Phylotype of Pseudomonas syringae Causing Bacterial Leaf Blight of Cantaloupe in China.

Bacterial leaf blight (BLB) has caused severe yield losses in cantaloupe (Cucumis melo L.) in the major melon-growing regions of China since the beginning of the twentieth century. Historically, Pseudomonas syringae pv. lachrymans was considered to be the causal agent of BLB of cantaloupe and angular leaf spot of cucumber. In the process of characterizing bacteria isolated from cantaloupe, we observed that putative P. syringae pv. lachrymans yielded negative results in P. syringae pv. lachrymans-specific PCR assays. This suggested that the P. syringae pv. lachrymans-like strains from cantaloupe were distinct from those recovered from cucumber. To investigate the differences between P. syringae pv. lachrymans-like strains isolated from cantaloupe and cucumber, 13 P. syringae strains isolated from cantaloupe [12 from China and 1 from Zimbabwe (NCPPB2916)] and 7 additional P. syringae reference strains were analyzed by catabolic profiling, phylogenetic analysis by multilocus sequence analysis (MLSA) and pathogenicity tests on cantaloupe leaflets. Catabolic profiling and MLSA based on 10 housekeeping genes and 2 hypersensitive response and pathogenicity (hrp) genes allowed us to differentiate strains isolated from cantaloupe and cucumber. Pseudomonas syringae pv. lachrymans strains isolated from cucumber clustered with genomospecies 2, and 13 P. syringae strains isolated from cantaloupe belonged to genomospecies 1. While all cantaloupe strains were closely related to P. syringae pv. aptata, they could be differentiated from this pathovar based on metabolic tests and MLSA. Pathogenicity tests showed that all strains isolated from cantaloupe and cucumber were only pathogenic on their original hosts. Based on these observations we conclude that P. syringae pv. lachrymans strains recovered from cantaloupe in China represent a novel phylotype.

A Proposal for a Genome Similarity-Based Taxonomy for Plant-Pathogenic Bacteria that Is Sufficiently Precise to Reflect Phylogeny, Host Range, and Outbreak Affiliation Applied to Pseudomonas syringae sensu lato as a Proof of Concept.

Taxonomy of plant pathogenic bacteria is challenging because pathogens of different crops often belong to the same named species but current taxonomy does not provide names for bacteria below the subspecies level. The introduction of the host range-based pathovar system in the 1980s provided a temporary solution to this problem but has many limitations. The affordability of genome sequencing now provides the opportunity for developing a new genome-based taxonomic framework. We already proposed to name individual bacterial isolates based on pairwise genome similarity. Here, we expand on this idea and propose to use genome similarity-based codes, which we now call life identification numbers (LINs), to describe and name bacterial taxa. Using 93 genomes of Pseudomonas syringae sensu lato, LINs were compared with a P. syringae genome tree whereby the assigned LINs were found to be informative of a majority of phylogenetic relationships. LINs also reflected host range and outbreak association for strains of P. syringae pathovar actinidiae, a pathovar for which many genome sequences are available. We conclude that LINs could provide the basis for a new taxonomic framework to address the shortcomings of the current pathovar system and to complement the current taxonomic system of bacteria in general.

Draft genome sequences of five Pseudomonas syringae pv. actinidifoliorum strains isolated in France

ABSTRACT Pseudomonas syringae pv. actinidifoliorum causes necrotic spots on the leaves of Actinidia deliciosa and Actinidia chinensis. P. syringae pv. actinidifoliorum has been detected in New Zealand, Australia, France and Spain. Four lineages were previously identified within the P. syringae pv. actinidifoliorum species group. Here, we report the draft genome sequences of five strains of P. syringae pv. actinidifoliorum representative of lineages 1, 2 and 4, isolated in France. The whole genomes of strains isolated in New Zealand, representative of P. syringae pv. actinidifoliorum lineages 1 and 3, were previously sequenced. The availability of supplementary P. syringae pv. actinidifoliorum genome sequences will be useful for developing molecular tools for pathogen detection and for performing comparative genomic analyses to study the relationship between P. syringae pv. actinidifoliorum and other kiwifruit pathogens, such as P. syringae pv. actinidiae.

Draft genome sequences of five Pseudomonas syringae pv. actinidifoliorum strains isolated in France.

Pseudomonas syringae pv. actinidifoliorum causes necrotic spots on the leaves of Actinidia deliciosa and Actinidia chinensis. P. syringae pv. actinidifoliorum has been detected in New Zealand, Australia, France and Spain. Four lineages were previously identified within the P. syringae pv. actinidifoliorum species group. Here, we report the draft genome sequences of five strains of P. syringae pv. actinidifoliorum representative of lineages 1, 2 and 4, isolated in France. The whole genomes of strains isolated in New Zealand, representative of P. syringae pv. actinidifoliorum lineages 1 and 3, were previously sequenced. The availability of supplementary P. syringae pv. actinidifoliorum genome sequences will be useful for developing molecular tools for pathogen detection and for performing comparative genomic analyses to study the relationship between P. syringae pv. actinidifoliorum and other kiwifruit pathogens, such as P. syringae pv. actinidiae.

Divorcing Strain Classification from Species Names.

Confusion about strain classification and nomenclature permeates modern microbiology. Although taxonomists have traditionally acted as gatekeepers of order, the numbers of, and speed at which, new strains are identified has outpaced the opportunity for professional classification for many lineages. Furthermore, the growth of bioinformatics and database-fueled investigations have placed metadata curation in the hands of researchers with little taxonomic experience. Here I describe practical challenges facing modern microbial taxonomy, provide an overview of complexities of classification for environmentally ubiquitous taxa like Pseudomonas syringae, and emphasize that classification can be independent of nomenclature. A move toward implementation of relational classification schemes based on inherent properties of whole genomes could provide sorely needed continuity in how strains are referenced across manuscripts and data sets.

Extensive Field Survey, Laboratory and Greenhouse Studies Reveal Complex Nature of Pseudomonas syringae-Associated Hazelnut Decline in Central Italy.

Pseudomonas avellanae (Pav) has been reported as the causal agent of bacterial decline and bacterial canker of hazelnut in Italy and Greece, respectively. Both hazelnut diseases were reported to be similar in terms of symptoms, severity and persistence. In this study, we found that both symptomatic and asymptomatic trees in the field were colonized by Pav. Multilocus Sequence Typing (MLST) analysis showed that Pav strains isolated during this study in Italy belong to the P. syringae phylogroup 1 and they are closely related to Pav strains previously isolated in Greece from hazelnut bacterial canker. On the other hand, strains isolated in earlier studies from hazelnut decline in Italy belong to both phylogroup 1 and 2 of P. syringae. Both phylogroup 1 strains of P. syringae from Greece and Italy are different than strains isolated in this study in terms of their capacity to excrete fluorescent pigments on different media. Despite the same plant genotype and cropping practices adopted, the incidence of hazelnut decline ranged from nearly 0 to 91% across our study sites. No disease developed on plants inoculated with Pav through wounding while leaf scar inoculations produced only mild disease symptoms. Based on our results and the previously reported correlation between pedo-climatic conditions and hazelnut decline, we conclude that hazelnut decline in central Italy could be incited by a combination of predisposing (adverse pedo-climatic conditions) and contributing factors (Pav). Because this is a true decline different from "bacterial canker" described in Greece, we refer to it as hazelnut decline (HD).

Genome analysis of the kiwifruit canker pathogen Pseudomonas syringae pv. actinidiae biovar 5.

Pseudomonas syringae pv. actinidiae (Psa) is a destructive pathogen of kiwifruit bacterial canker disease, causing severe economic losses to kiwifruit industry worldwide. Biovar 5 is the most recently reported biovar of Psa, and is found in only a local area of Japan at present. There is not much information of genetic characteristics of biovar 5. Thus, the genome of biovar 5 was sequenced and analyzed to clarify its detailed genetic characteristics. Here, the genomes of strain MAFF 212056 and MAFF 212061 of biovar 5 were estimated to be about 6.3 Mbp and 6.5 Mbp, respectively, and their phylogenetic positions were proved to be near that of biovar 2 in the phylogenetic tree. However, it was confirmed that biovar 5 had neither the coronatine biosynthetic genes conserved in biovar 2, its phylogenetic neighbor, nor the phaseolotoxin biosynthetic genes conserved in biovar 1, Japanese native pathogen. In addition, 45 genes of type III secreted effectors were identified in biovar 5 genomes, showing that their composition is different from that in the other biovars. Moreover, some biovar 5-specific regions were identified. Then, biovar 5-specific PCR primers for targeting these regions were designed, and proved to be applicable for detecting biovar 5 specifically.