Leaves and fruits of walnut trees exhibiting symptoms of bacterial blight were collected from six locations in Poland. Isolations on agar media resulted in 18 bacterial isolates with colony morphology resembling that of the Xanthomonas genus. PCR using X1 and X2 primers specific for Xanthomonas confirmed that all isolates belonged to this genus. In pathogenicity tests on unripe walnut fruits, all isolates caused typical black necrotic lesions covering almost the entire pericarp. Results of selected phenotypic tests indicated that characteristics of all isolates were the same as described for the type strain of Xanthomonas arboricola pv. juglandis. Genetic analyses (PCR MP, ERIC-, BOX-PCR and MLSA) showed similarities between the studied isolates and the reference strain of X. arboricola pv. juglandis CFBP 7179 originating from France. However, reference strains I-391 from Portugal and LMG 746 from the UK were different. MLSA analysis of partial sequences of the fyuA, gyrB and rpoD genes of studied isolates and respective sequences from GenBank of pathotype strains of other pathovars of X. arboricola showed that the X. arboricola pv. juglandis isolates consisted of different phylogenetic lineages. An incongruence among MLSA gene phylogenies and traces of intergenic recombination events were proved. These data suggest that the sequence analysis of several housekeeping genes is necessary for proper identification of X. arboricola pathovars.

Background Erwinia amylovora is generally considered to be a homogeneous species in terms of phenotypic and genetic features. However, strains show variation in their virulence, particularly on hosts with different susceptibility to fire blight. We applied the RNA-seq technique to elucidate transcriptome-level changes of the lowly virulent E. amylovora 650 strain during infection of shoots of susceptible (Idared) and resistant (Free Redstar) apple cultivars.ResultsThe highest number of differentially expressed E. amylovora genes between the two apple genotypes was observed at 24 h after inoculation. Six days after inoculation, only a few bacterial genes were differentially expressed in the susceptible and resistant apple cultivars. The analysis of differentially expressed gene functions showed that generally, higher expression of genes related to stress response and defence against toxic compounds was observed in Free Redstar. Also in this cultivar, higher expression of flagellar genes (FlaI), which are recognized as PAMP (pathogen-associated molecular pattern) by the innate immune systems of plants, was noted. Additionally, several genes that have not yet been proven to play a role in the pathogenic abilities of E. amylovora were found to be differentially expressed in the two apple cultivars.ConclusionsThis RNA-seq analysis generated a novel dataset describing the transcriptional response of the lowly virulent strain of E. amylovora in susceptible and resistant apple cultivar. Most genes were regulated in the same way in both apple cultivars, but there were also some cultivar-specific responses suggesting that the environment in Free Redstar is more stressful for bacteria what can be the reason of their inability to infect of this cultivar. Among genes with the highest fold change in expression between experimental combinations or with the highest transcript abundance, there are many genes without ascribed functions, which have never been tested for their role in pathogenicity. Overall, this study provides the first transcriptional profile by RNA-seq of E. amylovora during infection of a host plant and insights into the transcriptional response of this pathogen in the environments of susceptible and resistant apple plants.Electronic supplementary materialThe online version of this article (10.1186/s12864-017-4251-z) contains supplementary material, which is available to authorized users.

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Of thirty fluorescent Pseudomonas isolates originating from symptomatic tissues of sweet (Prunus avium) and sour cherry (Prunus cerasus), plum (Prunus domestica), peach (Prunus persica) and apricot (Prunus armeniaca), 23 were identified as P. syringae using LOPAT tests. Further characterization of those isolates by GATTa and L-lactate utilization tests showed that 10 of them belonged to race 1, six to race 2 of P. syringae pv. morsprunorum

To study the expression of pathogenicity-related genes in Erwinia amylovora, seven candidate reference genes (ffh, glyA, gyrA, proC, pykA, recA, rpoB) were selected and validated with the following five different mathematic algorithms: geNorm, NormFinder, BestKeeper, the delta CT method and the RefFinder web-based tool. An overall comprehensive ranking output from each of the selected software programs revealed that proC and recA, followed by ffh and pykA, were the most stably expressed genes and can be recommended for the normalization of RT-qPCR data. A combination of the three reference genes, proC, recA and ffh, allowed for the accurate expression analysis of amsB and hrpN genes and the calculation of their fold change in E. amylovora after its infection of susceptible and resistant apple cultivars. To the best of our knowledge, this is the first study presenting a list of the most suitable reference genes for use in the relative quantification of target gene expression in E. amylovora in planta, selected on the basis of a multi-algorithm analysis.