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

Abstract

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.

Characterization of Chilean Pseudomonas syringae pv actinidiae strains isolated from infected orchards

“…331 This can be seen in a Neighbor-Joining dendogram (Ciarroni et al 2015). Both results suggest a 332 high divergence within the PSA biovar 3 of the Chilean population, which could be explained by 333 the introduction of more than one biovar 3 strain or by the introduction of one strain that is in 334 constant change in order to better adapt to new environmental conditions.…”

Genetic diversity ofPseudomonas syringaepv.actinidiae, pathogen of kiwifruit bacterial canker

“…With regard to biovar 3, which has strong virulence and has become a major problem in various countries around the world, research using comparative genome analysis has been particularly intensively conducted. Based on the results, the biovar 3 strains currently distributed worldwide (hereafter referred to as ‘pandemic lineage’) were found to have been transmitted from China (Gallipoli et al ., ; Ciarroni et al ., ; McCann et al ., ; Vanneste, ). After biovar 3 strains had diversified over a long period of time in China, kiwifruit material, such as seedlings, scions and pollen, latently infected/contaminated with some strains of the pandemic lineage, have spread extensively due to exports from China that have increased rapidly in recent years, leading to the global spread of infection (pandemic) of Psa (Balestra et al ., ; Butler et al ., ; Cunty et al ., ; McCann et al ., ).…”

“…Thus, after integrating this information and the numerous research results obtained so far, phylogeographical analyses were conducted; as a result, concrete scenarios of the origin and evolution of Psa are becoming apparent, as described below (e.g. Ushiyama, 1993;Sawada et al, 1997Sawada et al, , 1999Sawada et al, , 2002Sawada et al, , 2014bGenka et al, 2006;Mazzaglia et al, 2012;Butler et al, 2013;McCann et al, 2013McCann et al, , 2017Vinatzer et al, 2014;Bartoli et al, 2015;Ciarroni et al, 2015;Cunty et al, 2015b;Fujikawa & Sawada, 2016Baltrus et al, 2017;Vanneste, 2017;He et al, 2019).…”

“…With regard to biovar 3, which has strong virulence and has become a major problem in various countries around the world, research using comparative genome analysis has been particularly intensively conducted. Based on the results, the biovar 3 strains currently distributed worldwide (hereafter referred to as 'pandemic lineage') were found to have been transmitted from China (Gallipoli et al, 2013;Ciarroni et al, 2015;McCann et al, 2017;Vanneste, 2017). After biovar 3 strains had diversified over a long period of time in China, kiwifruit material, such as seedlings, scions and Figure 3 A phylogenomic tree constructed based on the single nucleotide polymorphisms present in the core genome regions (core genome SNPs).…”

“…On the other hand, in addition to Japan, a small number of biovar 1 strains have also been detected in kiwifruit in Italy and Korea (a strain derived from Italy is indicated by the stars in Figures 2 and 3) (Mazzaglia et al, 2012;Scortichini et al, 2012;Cameron & Sarojini, 2014;Ciarroni et al, 2015;McCann et al, 2017). However, there is an opinion that these strains might have been transmitted from Japan (Ciarroni et al, 2015;Vanneste, 2017). In addition, there have been no reports that biovar 1 strains have been detected in countries other than Italy, Korea and Japan (Table 1).…”