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.
STUDY OF PATHOGENIC FACTORS OF E.COLI ISOLATED FROM PATIENTS WITH PERITONITIS
