Pseudomonas syringae pv. actinidiae (PSA) isolates from recent bacterial canker of kiwifruit outbreaks belong to the same genetic lineage

Abstract

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.

Figure 1. Phylogenetic tree placing P. syringae pv. actinidiae (PSA) within the P. syringae species complex.

1,186 proteins that are present exactly one time in each of the nine PSA strains and the P. syringae pv. theae pathotype strain NCPPB 2598 sequenced here and in each of 35 additional P. syringae strains for which genome sequences are available were aligned and concatenated. A maximum likelihood tree was then built using the two sequenced P. fluorescens strains Pf-0 and Pf5-1 as outgroups. Strains are labeled with pathovar names and strain names (genome accession numbers are listed in Table S1). 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.

Figure 2. Neighbor Joining cladogram based on Single Nucleotide Polymorphisms (SNPs) identified between P. syringae pv. actinidiae (PSA) genomes and P. syringae pv. theae.

Sequencing reads of nine PSA genomes were aligned against a draft genome of P. syringae pv. theae 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 P. syringae pv. theae 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.

Figure 3. Alignment of the genomic island PPHGI-1 from P. syringae pv phaseolicola 1302A

(Accession # AJ870974) with similar islands in PSA strains CFBP7286 (Italy), CH2010-6 (China), and MAFF302091 (Japan). Genomic regions of PSA strains were aligned with PPHGI-1 using BLASTN and visualized using the Artemis comparison tool . Positions of PCR products that distinguish the island present in strain CH2010-6 and MAFF302091 from the island present in strain CFBP7286 are indicated by two yellow and one orange rectangle respectively. content differences within the islands of strains CFBP7286 and CH2010-6 compared to the MAFF302091 genome are listed in Table S5.