Pseudomonas syringae pv. actinidiae (PSA) Isolates from Recent Bacterial Canker of Kiwifruit Outbreaks Belong to the Same Genetic Lineage

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

Number of SNPs distinguishing pairs of strains identified in 3,453,192 bp.

<p>Number of SNPs distinguishing pairs of strains identified in 3,453,192 bp.</p

Genome sequencing and assembly results for the nine sequenced PSA strains and the <i>P. syringae</i> pv. <i>theae</i> pathotype strain NCPPB 2598.

1<p>The versions described in this paper are the first versions xxxx01000000.</p>2<p>pathovar <i>theae</i>.</p

Phylogenetic tree placing <i>P. syringae</i> pv. <i>actinidiae</i> (PSA) within the <i>P. syringae</i> species complex.

<p>1,186 proteins that are present exactly one time in each of the nine PSA strains and the <i>P. syringae</i> pv. <i>theae</i> pathotype strain NCPPB 2598 sequenced here and in each of 35 additional <i>P. syringae</i> strains for which genome sequences are available were aligned and concatenated. A maximum likelihood tree was then built using the two sequenced <i>P. fluorescens</i> strains Pf-0 and Pf5-1 as outgroups. Strains are labeled with pathovar names and strain names (genome accession numbers are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036518#pone.0036518.s001" target="_blank">Table S1</a>). Bootstrap values higher than 95 are shown at nodes. * Strain ES4326 is present twice since two stocks of this strain were sequenced separately. ** Strains 0893-23 and NCPPB 3681 are the same strain sequenced twice in two separate genome sequencing projects.</p

Position of all identified SNPs in the <i>P. syringae</i> pv. <i>theae</i> NCPPB 2598 genome (and primer sequences to amplify them) that distinguish European strains from each other and European strains from Chinese strains. Primer sequences to distinguish between the European and Asian version of the PPHGI-1 island are also listed. Results obtained by PCR and Sanger sequencing are indicated.

<p>Position of all identified SNPs in the <i>P. syringae</i> pv. <i>theae</i> NCPPB 2598 genome (and primer sequences to amplify them) that distinguish European strains from each other and European strains from Chinese strains. Primer sequences to distinguish between the European and Asian version of the PPHGI-1 island are also listed. Results obtained by PCR and Sanger sequencing are indicated.</p

Neighbor Joining cladogram based on Single Nucleotide Polymorphisms (SNPs) identified between <i>P. syringae</i> pv. <i>actinidiae</i> (PSA) genomes and <i>P. syringae</i> pv. <i>theae</i>.

<p>Sequencing reads of nine PSA genomes were aligned against a draft genome of <i>P. syringae</i> pv. <i>theae</i> pathotype strain NCPPB 2598. A neighbor joining tree was built based on 21,494 SNPs so identified. Country and year of isolation are indicated for each strain. Bootstrap values based on 1000 bootstrap replicates are shown above nodes and number of SNPs compared to <i>P. syringae</i> pv. <i>theae</i> are shown underneath branches. Branches with less than 50% bootstrap support were collapsed. In the Japanese/Korean clade three SNPs group PsaKN.2 with PA459 and thus conflict with the branching pattern obtained in the tree. No SNPs conflict with the branching pattern obtained for the Chinese/European clade. A Bayesian tree was also constructed and had the same topology as the neighbor-joining tree.</p