Pseudomonas viridiflava, a Multi Host Plant Pathogen with Significant Genetic Variation at the Molecular Level

The pectinolytic species Pseudomonas viridiflava has a wide host range among plants, causing foliar and stem necrotic lesions and basal stem and root rots. However, little is known about the molecular evolution of this species. In this study we investigated the intraspecies genetic variation of P. viridiflava amongst local (Cretan), as well as international isolates of the pathogen. The genetic and phenotypic variability were investigated by molecular fingerprinting (rep-PCR) and partial sequencing of three housekeeping genes (gyrB, rpoD and rpoB), and by biochemical and pathogenicity profiling. The biochemical tests and pathogenicity profiling did not reveal any variability among the isolates studied. However, the molecular fingerprinting patterns and housekeeping gene sequences clearly differentiated them. In a broader phylogenetic comparison of housekeeping gene sequences deposited in GenBank, significant genetic variability at the molecular level was found between isolates of P. viridiflava originated from different host species as well as among isolates from the same host. Our results provide a basis for more comprehensive understanding of the biology, sources and shifts in genetic diversity and evolution of P. viridiflava populations and should support the development of molecular identification tools and epidemiological studies in diseases caused by this species

LOPAT tests of eighteen local (Crete, Greece) <i>P. viridiflava</i> isolates along with <i>P. viridiflava</i> reference strain NCPPB1249 and other pseudomonads.

<p>LOPAT tests of eighteen local (Crete, Greece) <i>P. viridiflava</i> isolates along with <i>P. viridiflava</i> reference strain NCPPB1249 and other pseudomonads.</p

Bacterial strains used in this study for biochemical characterization and pathogenicity tests.

<p>Bacterial strains used in this study for biochemical characterization and pathogenicity tests.</p

<i>P. viridiflava</i> phylogenetic trees, utilizing <i>rpoD</i> sequences along with sequences obtained from GenBank.

<p>The evolutionary history was inferred using the Neighbor-Joining method. Tree construction and evolutionary distances were carried out as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036090#pone-0036090-g002" target="_blank">Figure 2</a> legend. The analysis involved 32 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 513 positions in the final dataset. The methodology used for the evolutionary analysis, tree construction and other details are described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036090#pone-0036090-g003" target="_blank">Figure 3</a> legend.</p

<i>P. viridiflava</i> phylogenetic trees, utilizing <i>rpoB</i> sequences along with sequences obtained from GenBank.

<p>The evolutionary history was inferred using the Neighbor-Joining method. Tree construction and evolutionary distances were carried out as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036090#pone-0036090-g002" target="_blank">Figure 2</a> legend. The analysis involved 27 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 741 positions in the final dataset. The methodology used for the evolutionary analysis, tree construction and other details are described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036090#pone-0036090-g003" target="_blank">Figure 3</a> legend.</p

Agarose gel electrophoresis of BOX-PCR of 18 local <i>P. viridiflava</i> isolates.

<p>Agarose gel electrophoresis of BOX-PCR amplification products from genomic DNA of 18 local <i>P. viridiflava</i> isolates. The molecular size marker is λ phage DNA digested with the restriction endonuclease <i>Pst</i>I. The negative film filter was applied to the image of an ethidium bromide gel. Isolate codes are given over each lane.</p

Phylogenetic trees of the local <i>P. viridiflava</i> isolates.

<p>The construction of the dendrograms was based on <b>A</b>: BOX- and ERIC-PCR fingerprints (rep-PCR) and <b>B</b>: the combined <i>gyrB, rpoD</i> and <i>rpoB</i> gene sequences. The plant hosts are given next to the code number (PVXXX, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036090#pone-0036090-t001" target="_blank">Table 1</a>) of each isolate. The evolutionary history was inferred using the UPGMA method. The consensus tree inferred from 1500 replicates is taken to represent the evolutionary history of the isolates analyzed. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test is shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Maximum Composite Likelihood method and are in the units of the number of base substitutions per site. All positions containing gaps and missing data were eliminated. There were a total of 2222 positions in the final dataset. Evolutionary analyses were conducted in MEGA5.</p

<i>P. viridiflava</i> phylogenetic tree, utilizing <i>gyrB</i> sequences determined in this study along with sequences obtained from GenBank.

<p>The evolutionary history was inferred using the Neighbor-Joining method. The bootstrap consensus tree inferred from 1500 replicates is taken to represent the evolutionary history of the taxa analyzed. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test are shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Maximum Composite Likelihood method and are in the units of the number of base substitutions per site. The analysis involved 52 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 740 positions in the final dataset. Evolutionary analyses were conducted in MEGA5. The host plant species is presented next to the code number (e.g. PVXXX) of each isolate.</p

Local bacterial strains used in this study for <i>gyrB</i>, <i>rpoD</i> and <i>rpoB</i> phylogenetic analysis.

<p>Information for other strains used, can be found in supplementary <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036090#pone.0036090.s006" target="_blank">Table S3</a>.</p