Pseudomonas avellanae is the causal agent of hazelnut ( Corylus avellana ) decline, both in northern Greece and central Italy, and two lineages related to the geographical origins of the pathogen have previously been identified. Forty strains, obtained from all the areas where the disease has so far been observed, and representing six different subpopulations of the two lineages, were further assessed using insertion-sequence PCR genomic fingerprinting. The data previously obtained from repetitive-sequence PCR using ERIC and BOX primer sets and insertion-sequence PCR (IS50) were analysed using statistical methods, enabling genetic diversity and gene flow among the populations to be elucidated, as well as verifying the possible correlation between genetic diversity and geographical origin. The Mantel test performed with ERIC, BOX and IS50-PCR data revealed that the P. avellanae populations that are spatially distant from each other are also genetically dissimilar: gene flow estimates confirmed this. The present study supports the hypothesis that P. avellanae originated separately in Greece and Italy, and that the two lineages of the pathogen underwent separate local evolution.
To assess the genetic diversity and genetic relationships of Pseudomonas avellanae, the causative agent of hazelnut decline, a total of 102 strains, obtained from central Italy (provinces of Viterbo and Rome) and northern Greece, were studied using multilocus enzyme electrophoresis (MLEE). Their allelic variation in 10 loci was determined. All loci were polymorphic and 53 electrophoretic types (ETs) were identified from the total sample. The mean genetic diversity (H) was 0?65 and this value ranged from 0?37 for the least polymorphic to 0?82 for the most polymorphic locus. The dendrogram originated from MLEE data indicated two main groups of ETs, A and B. The groups do not appear to be correlated to the geographic origin of the strains, although all the ETs from northern Greece clustered into subgroup B1. Pseudomonas syringae pv. actinidiae and P. syringae pv. theae, included in the analysis as outgroups, clustered apart. The index of association (I A ) for P. avellanae was 0?90. The I A values were always significantly different from zero for the population subsets studied and no epidemic structure was found. These results would indicate that the population structure of P. avellanae is clonal either in northern Greece or in central Italy. The recent outbreaks of the bacterium in new areas of hazelnut cultivation would explain the current clonal structure that is persisting over decades.
The 16S-23S rRNA gene internal transcribed spacer region (ITS1) from 34 strains of Pseudomonas avellanae and some strains of Pseudomonas syringae pathovars was amplified and assessed by restriction fragment length polymorphism (RFLP) using 10 restriction enzymes. In addition, the ITS1 region of four representative P. avellanae strains was sequenced and compared by the neighbour-joining algorithm with that of P. syringae pathovars. Two main groups of P. avellanae strains were observed that did not correlate with their origin. The ITS1 region sequencing revealed a high similarity with the P. syringae complex. One group of P. avellanae strains showed high similarity to P. s. pv. actinidiae and P. s. pv. tomato; another group showed similarity with P. s. pv. tabaci and P. s. pv. glycinea. Two strains clustered with P. s. pv. pisi. The difficulties to unambiguously classify the strains associated with hazelnut decline in Greece and Italy are discussed.
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