Pseudomonas syringae pv. actinidiae draft genomes comparison reveal strain-specific features involved in adaptation and virulence to Actinidia species

Abstract

A recent re-emerging bacterial canker disease incited by Pseudomonas syringae pv. actinidiae (Psa) is causing severe economic losses to Actinidia chinensis and A. deliciosa cultivations in southern Europe, New Zealand, Chile and South Korea. Little is known about the genetic features of this pathovar. We generated genome-wide Illumina sequence data from two Psa strains causing outbreaks of bacterial canker on the A. deliciosa cv. Hayward in Japan (J-Psa, type-strain of the pathovar) and in Italy (I-Psa) in 1984 and 1992, respectively as well as from a Psa strain (I2-Psa) isolated at the beginning of the recent epidemic on A. chinensis cv. Hort16A in Italy. All strains were isolated from typical leaf spot symptoms. The phylogenetic relationships revealed that Psa is more closely related to P. s. pv. theae than to P. avellanae within genomospecies 8. Comparative genomic analyses revealed both relevant intrapathovar variations and putative pathovar-specific genomic regions in Psa. The genomic sequences of J-Psa and I-Psa were very similar. Conversely, the I2-Psa genome encodes four additional effector protein genes, lacks a 50 kb plasmid and the phaseolotoxin gene cluster, argK-tox but has acquired a 160 kb plasmid and putative prophage sequences. Several lines of evidence from the analysis of the genome sequences support the hypothesis that this strain did not evolve from the Psa population that caused the epidemics in 1984-1992 in Japan and Italy but rather is the product of a recent independent evolution of the pathovar actinidiae for infecting Actinidia spp. All Psa strains share the genetic potential for copper resistance, antibiotic detoxification, high affinity iron acquisition and detoxification of nitric oxide of plant origin. Similar to other sequenced phytopathogenic pseudomonads associated with woody plant species, the Psa strains isolated from leaves also display a set of genes involved in the catabolism of plant-derived aromatic compounds.

Figure 1. Disease symptoms of Psa on Actinidia spp. leaves and main leader.

The sequenced I-Psa and I2-Psa strains from Italy were isolated from the leaves herein showed. A) Leaf symptoms on Actinidia deliciosa cv. Hayward (June, 1992); b) leaf symptom on A. chinensis cv. Hort16A (June, 2008). Note the red-rusty colour of the spots and the chlorotic halo on A. deliciosa and the brownish spot without halo on A. chinensis; c) large canker in deep winter, induced by Psa on the main leader of an adult A. chinensis cv. Hort16A plant, in central Italy (February, 2009). Note the complete destruction of all the external woody tissues of the plant.

Figure 2. Pairwise alignment between the draft genomes of J-Psa, I-Psa and I2-Psa and the complete genome of P. s. pv. tomatoDC3000 using the MAUVE software.

Colored blocks outline genome sequence that align to part of another genome, and is presumably homologous and internally free from genomic rearrangement (Locally Colinear Blocks or LCBs). Areas that are completely white were not aligned and probably contain sequence elements specific to a particular genome. Blocks below the centre line indicate regions that align in the reverse complement (inverse) orientation. A profile is drawn within each LCB with the height of the color corresponding to the average degree of sequence conservation.

Figure 3. Representative part of the genome alignment between Psa strains and Pto DC3000 showing some variable regions.

The violet segments (on the right) point out the variable region 3, present in all three Psa strains but not in Pto DC3000; the deep blue (on the left) segments point out the variable region 2, present in J-Psa and I-Psa but absent in I2-Psa and Pto DC3000. The blue segments indicate another variable region present in Pto DC3000 and I2-Psa but not in J-Psa and I2-Psa. The figure shows also some others shorter regions (i.e. light green segments) probable examples of horizontal gene transfer.

Figure 4. Evolutionary relationships of Psa strains to other phytopathogenic pseudomonads.

Phylogenetic relationships were estimated from concatenated sequences from three housekeeping genes, gyrB, rpoB and rpoD (1,646 bp), using the neighbour-joining (NJ) algorithm. Bootstrap values are reported at each branching. Members of all nine genomospecies (Gardan et al., 1999), except genomospecies 7, are included into the analysis. The letter followed by the number reported in brackets indicates the genomospecies sensu Gardan et al. .

Figure 5. Genealogy of strains of genomospecies 8.

(A) Maximum likelihood tree resulting from the analysis of a concatenation of 171 ORFs. (B) An alternative genealogical hypothesis with the constrain of the common origin of the Italian Psa strains.

Figure 6. Venn diagram of the type III effector gene complements of J-Psa, I-Psa and I2-Psa strains based on the comparison of the same complement of other sequenced plant pathogenic pseudomonads.

The genes conserved among the three strains are indicated in the middle of the diagram. J-Psa and I-Psa as well as I2-Psa display four unique different effector genes (see also Table S3).

Figure 7. Plasmid profiles of Psa.

Agarose gel electrophoresis to compare the number and size of native plasmids in the genome of Psa strains. The gels show also other representative Psa strains from the outbreak of bacterial canker in Japan (i.e. 1984) and from the current severe epidemics in Italy. See also Materials and Methods. Note as the ca. 50 kb plasmid present in J-Psa and I-Psa is not contained in all the Psa strains isolated from the current epidemic in Italy. By contrast, I2-Psa and other strains obtained from the recent epidemics of bacterial canker in Italy contain a plasmid of about 160 kb.

Figure 8. Multiplication trends of Psa strains in Actinidia species.

Multiplication in A. deliciosa cv Hayward (a) and in A. chinensis cv Hort16A (b) leaves. Bacteria were inoculated at 1–2×10³ and 1–2×10⁶ cfu/ml. Data represent the mean log of bacterial cell number and standard deviation (SD) as obtained from eight inoculation sites per each sample.