A study was performed in order to assess the presence of Xylella fastidiosa in imported ornamental plants, among them Olea europaea, Coffea arabica and Nerium oleander. Positive results were only obtained from C. arabica, where 15 plant samples tested positive for X. fastidiosa by PCR, nine from Costa Rica and six from Honduras. Transmission electron microscopy observations indicated that rod‐shaped bacterial cells exhibiting the characteristics of X. fastidiosa cells were present in the xylem vessels of leaf petioles obtained from the infected C. arabica plants. Diversity of X. fastidiosa in C. arabica plants was assessed through a multilocus sequence typing (MLST) analysis of seven housekeeping genes (leuA, petC, lacF, cysG, holC, nuoL and gltT) and compared with X. fastidiosa infecting different host plants worldwide. Based on this MLST analysis, the prevalence of different sequence types (STs) of X. fastidiosa in the C. arabica ornamental plants was demonstrated and related to different X. fastidiosa subspecies, underlining the risk of introducing additional genetic diversity for X. fastidiosa to Europe. ST53, related to X. fastidiosa subsp. pauca, was frequently found in these C. arabica samples. A second ST related to X. fastidiosa subsp. pauca, ST73, has been assessed in coinfection with ST53 in one individual plant. Additionally, ST72 and ST76, related to X. fastidiosa subsp. fastidiosa, have been recorded. Next to these previously described STs, a novel ST, namely ST77 has been revealed, related to X. fastidiosa subsp. fastidiosa. Isolation of X. fastidiosa from leaf petioles and midribs of infected C. arabica plants was successfully performed only after the application of an additional ultrasonication step during the extraction procedure. Based on this approach, a number of X. fastidiosa isolates were obtained and further characterized.

During the last two years, greenhouse cultivation of rose (Rosa spp.) in the Netherlands has been challenged by an uncommon bacterial disease. Affected plants suffered from chlorosis, stunting, wilting, and necrosis. The bacterial isolates obtained from the different Rosa spp. cultivars were all identified as phylotype I, sequevar 33 from the 'Ralstonia solanacearum species complex' (RSSC), actually reclassified as Ralstonia pseudosolanacearum. The work in this paper considers the genetic diversity and the phylogenetic position of 129 R. pseudosolanacearum isolates from the outbreak. This was assessed by AFLP based on four different primer combinations and MLP using partial sequences of the egl, mutS, and fliC genes. The AFLP revealed identical profiles for all the isolates, irrespective of their association with Rosa sp. propagating material, Rosa spp. plants for cut flowers, or water used in the different greenhouse cultivations. These AFLP profiles were unique and diverged from profiles of all other reference isolates in the RSSC included. Furthermore, MLP on egl, fliC, and mutS gene sequences clearly demonstrated that all R. pseudosolanacearum isolates clustered in phylotype I, as a distinct monophyletic group. Interestingly, this monophyletic group also included phylotype I strain Rs-09-161 from eggplant (Solanum melongena), isolated in 2009 in India. AFLP and MLP were both efficient in revealing the genetic divergence from the RSSC isolates included. The phylogenetic tree constructed from the AFLP profiles was, in general, in agreement with the one obtained from MLP. Both phylogenetic trees displayed a similar clustering, supported by high posterior probabilities. Both methodologies clearly demonstrated that the R. pseudosolanacearum isolates from Rosa spp. grouped in a monophyletic group inside phylotype I, with a particular correspondence to a strain present in India, as revealed in MLP.

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In 2008, Dutch ornamental plant growers observed a leaf spot of cherry laurel (Prunus laurocerasus) at a greater incidence (5 to 50%) than the usual sporadic level (<1%). For advice on disease control, ~5 to 10% of these growers contacted Dutch regulatory officials. In November and December 2008, six symptomatic samples from northern and southern parts of the Netherlands were submitted for diagnosis. Leaf spots were chlorotic, most had a necrotic brown center with a distinct margin, and the spots readily abscised, resulting in a “shot-hole” appearance. Leaf spots from the samples were surface sterilized (2 s in 70% vol/vol alcohol), blotted dry on tissue paper, chopped into pieces (1 to 2 mm in diameter), and incubated for 30 min in 10 mM phosphate-buffered saline (PBS) (1). A 20-μl aliquot of extract per sample was streaked by dilution plating on four plates of yeast peptone glucose agar medium (1), and the plates were incubated for 2 to 3 days at 28°C. Isolations from all six samples yielded Xanthomonas-like colonies. After purification, characterization of all six isolates revealed oxidative, nonfermentative metabolism of glucose by rod-shaped, gram-negative bacterial cells. All six isolates were identified as Xanthomonas arboricola pv. pruni based on biochemical tests (1), fatty acid analysis (4), and immunofluorescence (IF) using polyclonal antibodies (Plant Research International, the Netherlands). Pathogenicity was tested on potted peach plants (cvs. Peregrine and Vaes Oogst) and on detached leaves of P. laurocerasus (cv. Novita) (1). The six field isolates from 2008 were each inoculated (108 CFU/ml) onto four leaves per plant of each of two peach plants (replicates). As positive control treatments, two reference strains (ATCC 19312 and PD740) were each inoculated onto the same number of leaves and plants, and as a noninoculated negative control treatment, leaves of two peach plants were treated with sterile 10 mM PBS buffer (1). All leaves inoculated with the six field isolates and the two reference strains developed typical bacterial spot symptoms in 3 to 4 weeks. Negative control plants showed no symptoms. The detached leaf assay performed with the same treatments on each of two leaves (replicates) showed identical results. The bacterium was reisolated from leaf spots associated with each of the eight symptomatic treatments and identity of the reisolates was confirmed by IF. Additionally, genotypic variation of 35 Dutch isolates of X. arboricola pv. pruni was assessed by BOX-PCR assay with the BOX A1R primer set (3), and Gyrase B gene sequencing (2). Both methods revealed 100% homology among the 35 isolates, suggesting a single, recent introduction of X. arboricola pv. pruni into the Netherlands. In a 2009 survey to assess distribution of the disease in the Netherlands, X. arboricola pv. pruni was found in 41 fields. Infected hosts included P. laurocerasus cvs. Otto Luyken, Rotundifolia, Novita, Etna, Anbri, Herbergii, Mischeana, and Caucasia. X. arboricola pv. pruni is a quarantine organism in countries affiliated under the EPPO (European and Mediterranean Plant Protection Organization). Phytosanitary measures were taken to prevent movement of infested plants from nurseries where X. arboricola pv. pruni was detected. References: (1) Anonymous. EPPO Bull. 36:129, 2006. (2) N. Parkinson et al. Int. J. Syst. Evol. Microbiol. 59:264, 2009. (3) J. Versalovic et al. Methods Mol. Cell. Biol. 5:25, 1994. (4) S. A. Weller et al. EPPO Bull. 30:375, 2000.