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

Development of a Multiple Loci Variable Number of Tandem Repeats Analysis (MLVA) to Unravel the Intra-Pathovar Structure of Pseudomonas syringae pv. actinidiae Populations Worldwide.

The bacterial canker of kiwifruit by Pseudomonas syringae pv. actinidiae is an emblematic example of a catastrophic disease of fruit crops. In 2008 a new, extremely virulent form of the pathogen emerged and rapidly devastated many Actinidia spp. orchards all over the world. In order to understand differences in populations within this pathovar and to elucidate their diffusion and movements on world scale, it is necessary to be able to quickly and on a routine basis compare new isolates with previous records. In this report a worldwide collection of 142 strains was analyzed by MLVA, chosen as investigative technique for its efficacy, reproducibility, simplicity and low cost. A panel of 13 Variable Number of Tandem Repeats (VNTR) loci was identified and used to describe the pathogen population. The MLVA clustering is highly congruent with the population structure as previously established by other molecular approaches including whole genome sequencing and correlates with geographic origin, time of isolation and virulence. For convenience, we divided the VNTR loci in two panels. Panel 1 assay, using six loci, recognizes 23 different haplotypes, clustered into ten complexes with highest congruence with previous classifications. Panel 2, with seven VNTR loci, provides discriminatory power. Using the total set of 13 VNTR loci, 58 haplotypes can be distinguished. The recent hypervirulent type shows very limited diversity and includes, beside the strains from Europe, New Zealand and Chile, a few strains from Shaanxi, China. A broad genetic variability is observed in China, but different types are also retrievable in Japan and Korea. The low virulent strains cluster together and are very different from the other MLVA genotypes. Data were used to generate a public database in MLVAbank. MLVA represents a very promising first-line assay for large-scale routine genotyping, prior to whole genome sequencing of only the most relevant samples

Unweighted Neighbor-Joining dendrogram of the strains from China.

<p>The UNJ dendrogram represents results from the analysis of the fifty-two strains enclosed in the “China” group (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135310#pone.0135310.g003" target="_blank">Fig 3</a>) with the complete set of 13 VNTR loci. Numbers on branches represents the percentage of bootstraps on 500 replications, when higher than 60%.</p

Comparison of dendrograms obtained by SNPs and MLVA analysis.

<p>The dendrogram on the left was obtained by Maximum Likelihood analysis on 15,329 SNPs from WGS sequences of 28 Psa strains [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135310#pone.0135310.ref037" target="_blank">37</a>], whilst the radial dendrogram on the right was obtained by Unweighted Neighbor Joining tree from six selected VNTR loci (Panel 1) on 20 strains used in this study selected for having geographical and time origins identical or corresponding to the first ones. Numbers on branches represents the percentage of bootstraps on 500 replications.</p

Minimum spanning tree of 142 Psa strains obtained by MLVA6—Panel 1.

<p>The MST was obtained by Bionumerics 7.5 analyzing the whole complex of strains on the basis of the six Panel 1 VNTR loci. Each circle represents a group of strains with identical MLVA haplotype and its dimension is proportional to the number of strains included. The grey shaded areas represent the clonal complexes (CC), defined as groups of strains connected with maximum branch length of one. The CCs and singletons circumscribed by dashed lines enclose strains of the main geographical groups: China, Japan and Korea. The red circles enclose the strains that engendered the former definition of 4 Psa biovars.</p