Pseudomonas syringae pv. actinidiae from recent outbreaks of kiwifruit bacterial canker belong to different clones that originated in China

Abstract

A recently emerged plant disease, bacterial canker of kiwifruit (Actinidia deliciosa and A. chinensis), is caused by Pseudomonas syringae pv. actinidiae (PSA). The disease was first reported in China and Japan in the 1980s. A severe outbreak of PSA began in Italy in 2008 and has spread to other European countries. PSA was found in both New Zealand and Chile in 2010. To study the evolution of the pathogen and analyse the transmission of PSA between countries, genomes of strains from China and Japan (where the genus Actinidia is endemic), Italy, New Zealand and Chile were sequenced. The genomes of PSA strains are very similar. However, all strains from New Zealand share several single nucleotide polymorphisms (SNPs) that distinguish them from all other PSA strains. Similarly, all the PSA strains from the 2008 Italian outbreak form a distinct clonal group and those from Chile form a third group. In addition to the rare SNPs present in the core genomes, there is abundant genetic diversity in a genomic island that is part of the accessory genome. The island from several Chinese strains is almost identical to the island present in the New Zealand strains. The island from a different Chinese strain is identical to the island present in the strains from the recent Italian outbreak. The Chilean strains of PSA carry a third variant of this island. These genomic islands are integrative conjugative elements (ICEs). Sequencing of these ICEs provides evidence of three recent horizontal transmissions of ICE from other strains of Pseudomonas syringae to PSA. The analyses of the core genome SNPs and the ICEs, combined with disease history, all support the hypothesis of an independent Chinese origin for both the Italian and the New Zealand outbreaks and suggest the Chilean strains also originate from China.

Figure 1. Evolutionary relationships of P. syringae pv. actinidiae and members of the P. syringae species complex.

Phylogenetic tree constructed using concatenated sequences of four core genome loci (gapdh, gltA, gyrB and rpoD), with bootstrap values greater than 75 per cent (1050 replicates) shown at the nodes. The evolutionary history was inferred using the Neighbor-Joining method in MEGA5 . The PSA strains are enclosed by a blue shaded box, with their strain number and country of origin shown. PsD strains are enclosed by an orange shaded box and PsHa strains are enclosed by a green shaded box. The strain numbers of other members of the P. syringae complex are also shown.

Figure 2. Comparison of the island in P. syringae pv. syringae B728a (PsyrGI-6) with those in PSA.

Artemis Comparison Tool alignments of PsyrGI-6 with (a) the island in ICMP18708 (NZ) (Pac_ICE1), (b) ICMP18744 (Italy)(Pac_ICE2) and (c) ICMP19455 (Chile)(Pac_ICE3). The blue areas refer to regions that are inverted between islands. Darker red regions are those that share the highest similarity.

Figure 3. Comparative alignments of the islands in PSA.

A. Comparison of Pac_ICEs from New Zealand and Italian PSA with Pac_ICEs from Chinese PSA strains. Upper panel: alignment of Pac_ICE1 from strain M7 (China) and ICMP18708 (New Zealand). Middle panel: alignment of Pac_ICE1 from ICMP18708 (NZ) and Pac_ICE2 from ICMP18744 (Italy). Lower panel: alignment of Pac_ICE2 from ICMP18744 (Italy) and M228 (China). B. Comparisons made with Pac_ICE3 from PSA from Chile. Upper panel: Comparison of Pac_ICE3 from ICMP19455 (Chile) with Pac_ICE1 from ICMP18708 (New Zealand). Lower panel: alignment of Pac_ICE3 from ICMP19455 (Chile) with that of Pac_ICE2 from ICMP18744 (Italy). The blue areas denote where two regions are inverted with respect to one another.

Figure 4. Mauve alignment of the genomic islands of PSA and P. syringae pv. syringae B728a.

Depiction of Pac_ICE3 in ICMP19455 (Chile), PsyGI-6 in P. syringae pv. syringae B728a, Pac_ICE2 (ICMP18744, Italy) and Pac_ICE1 (ICMP18708, NZ) using Mauve . Colored blocks each represent a locally co-linear block containing no apparent re-arrangements. Blocks below the centre line indicate regions that align in the reverse complement (inverse) orientation. Areas that are completely white were not aligned and probably contain sequence elements specific to a particular island. The height of the colour bars represents the average degree of sequence similarity.

Figure 5. Depiction of mobile elements within the ICEs of PSA and P. syringae pv. avellanae.

ICEs represented are Pac_ICE1 (top), present in PSA strains from New Zealand and in M7 (China); the ICE in P. syringae pv. avellanae Ve013; Pac_ICE2, present in PSA strains from Italy and in M228 (China) and Pac_ICE3, present in strains from Chile. Orange boxes indicate the ∼16 kb XerC transposon, Tn6212, present at almost 100% identity in the four ICEs shown and present in several ICEs in other P. syringae isolates/pathovars. Tn6211 is represented in PSA ICEs by a green box and an arrow showing the orientation of the three ORFs (including a methyl-accepting chemotaxis protein gene) present in these transposons. Tn6213 is an homologous element in P. syringae pv. avellanae, with ∼99% sequence identity to Tn6211. The red bars represent an IS element (1665 bp) which contains two overlapping ORFs with similarity to the transposases of IS3/IS911 family IS elements. It is present in only two of the ICEs examined so far, Pac_ICE1-nz in strain 6-1 at position1 and in Pac_ICE1-cn (in M7) at position2.

Figure 6. Evidence of Pac_ICE excision from the PSA genome.

PCR was carried out with genomic DNA from ICMP18708 (lanes 2–4) or from ICMP18744 (lanes 5–7). Lane 1: molecular weight markers. Lanes 2 and 5: amplicons generated with internal ICE primers facing out into the genomic context. Lanes 3 and 6: amplicons generated using primers in the genomic context flanking each Pac_ICE. Lanes 4: amplicon generated using primers flanking the lysine tRNA gene near exsB (unoccupied in ICMP18708). Lane 7: amplicon generated using primers flanking the lysine tRNA gene near ClpB (unoccupied in ICMP18744). Lane 8∶100 bp molecular weight ladder.

Figure 7. Evolutionary relationships of ICEs within strains of the P. syringae complex.

The concatenated sequences of seven proteins (XerC/D tyrosine recombinase, ICE_TraI, DSBA oxoreductase, uvrD helicase, AdoMet-methyl transferase, DnaB and ParA (chromosome partitioning) encoded in the ICEs were used to generate the phylogenetic tree. The data for islands in strains other than those sequenced here were obtained from the accession numbers shown in Table 7. The evolutionary history was inferred using the Neighbour-Joining method in MEGA5 . The optimal tree with the sum of branch length = 0.542 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1050 replicates) is shown next to the branches.