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BackgroundXanthomonas fragariae (Xf) is a bacterial strawberry pathogen and an A2 quarantine organism on strawberry planting stock in the EU. It is taxonomically and metabolically distinct within the genus Xanthomonas, and known for its host specificity. As part of a broader pathogenicity study, the genome of a Belgian, virulent Xf strain (LMG 25863) was assembled to draft status and examined for its pathogenicity related gene content.ResultsThe Xf draft genome (4.2 Mb) was considerably smaller than most known Xanthomonas genomes (~5 Mb). Only half of the genes coding for TonB-dependent transporters and cell-wall degrading enzymes that are typically present in other Xanthomonas genomes, were found in Xf. Other missing genes/regions with a possible impact on its plant-host interaction were: i) the three loci for xylan degradation and metabolism, ii) a locus coding for a ß-ketoadipate phenolics catabolism pathway, iii) xcs, one of two Type II Secretion System coding regions in Xanthomonas, and iv) the genes coding for the glyoxylate shunt pathway. Conversely, the Xf genome revealed a high content of externally derived DNA and several uncommon, possibly virulence-related features: a Type VI Secretion System, a second Type IV Secretion System and a distinct Type III Secretion System effector repertoire comprised of multiple rare effectors and several putative new ones.ConclusionsThe draft genome sequence of LMG 25863 confirms the distinct phylogenetic position of Xf within the genus Xanthomonas and reveals a patchwork of both lost and newly acquired genomic features. These features may help explain the specific, mostly endophytic association of Xf with the strawberry plant.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-14-829) contains supplementary material, which is available to authorized users.

Polyphasic analysis exposed important heterogeneity between bacterial strains catalogued as Xanthomonas arboricola pv. fragariae (Xaf) from different culture collections. Two draft whole-genome sequences revealed pathogenicity related genes of the type-three secretion system in strain LMG 19146, whereas none were found in the Xaf pathotype strain LMG 19145. Also, considerable sequence divergence was observed in the phylogenetic marker genes gyrB, rpoD, dnaK and fyuA. Further study of 16 Xaf culture-collection strains showed that co-classification is not justified. Partial 16S rRNA gene and gyrB sequencing demonstrated that 12 strains belonged to X. arboricola, but that they did not form one homogeneous group within the species. The four remaining strains were identified as Xanthomonas fragariae and Xanthomonas sp. All sequence-based identifications were confirmed by MALDI-TOF MS fingerprinting. Also, the pathogenicity genes hrcQ and avrBs2 were detected in only three of the 12 analysed X. arboricola strains. The X. arboricola and Xanthomonas sp. strains showed pectolytic activity, and upon inoculation in strawberry none of the strains reproduced the leaf blight symptoms reported for Xaf. This study demonstrates that (i) no clear criteria exist for the identification of strains as Xaf, (ii) the name Xaf is currently used for a genetically diverse assortment of strains, and (iii) the species X. arboricola holds many undetermined plant-associated bacteria besides the described pathovars.

Xanthomonas fragariae , the causal agent of angular leaf spot on strawberry, is a quarantine organism in strawberry propagation material in the European Union. For the reliable screening of planting material for latent infections, a realtime PCR assay based on Taqman® chemistry for the detection of X. fragariae was developed. Primers and probe sequences were based on a DNA fragment amplified by a previously reported X. fragariae -specific technique. The sequence of this genomic fragment had no significant similarity with any published GenBank sequence. Specificity of the designed assay was tested with an extended range of X. fragariae collection strains and isolates, with other Xanthomonas spp. and with unidentified bacterial isolates from strawberry plants. A nested PCR, which until now was the reference method for sensitive detection in planta , cross-reacted with the reference strain of Xanthomonas campestris pv. campestris . In combination with an elaborated DNA extraction procedure, the Taqman® PCR enabled reliable detection down to 300 colony forming units in a 100 mg strawberry leaf sample. The assay offers a new tool for epidemiological research and for sanitary control of plant material with low level or latent infections of X. fragariae .

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