First report of bacterial leaf spot of Hydrangea in retail nurseries in Belgium caused by strains assigned to a new Xanthomonas hortorum clade

The genus Hydrangea includes at least 23 species of ornamental plants that are highly valued for their large and long-lasting flowerheads. Incidents of leaf spot were regularly observed on Hydrangea arborescens and H. quercifolia in retail nurseries in Flanders (Belgium) from 2011 to 2015. Leaves showed brown to black irregular leaf spots (Fig. 1) which tended to blend into larger lesions (Fig. 2). Leaf spots were aseptically excised and comminuted in sterile 10 mM phosphate buffer (PB, pH 7.0). Dilution plating was done on BD DifcoTM Pseudomonas Agar F supplemented with sucrose (10 g/L). Large numbers of pale yellow round, convex Xanthomonas-like colonies had developed after four days incubation at 28°C. They induced a hypersensitive reaction after infiltration in leaves of tomato and Sedum, but not in leaves of tobacco (cv. Xanthi NN). The phylogenetic position in the genus Xanthomonas was determined with partial sequences of the DNA gyrase subunit B (gyrB) gene (Constantin et al., 2016), which were trimmed, aligned and compared with gyrB sequences of Xanthomonas benchmark strains in our Bionumerics database (Applied Maths, Bio-Mérieux) or retrieved from GenBank (NZ_SMED00000000.1 from Xanthomonas vitians LMG 938neoPT). GenBank accession numbers of the partial gyrB sequences of Xanthomonas isolates obtained in this study are shown in Table 1. The Hydrangea leaf spot isolates were assigned to a new clade in the gyrB-based phylogeny (Fig. 3 ) of X. hortorum and clearly distinct from any of the existing pathovars in the species (Morinière et al., 2020). Further analysis using whole genome sequences and ANI values is ongoing to determine whether it should be considered a new Xanthomonas species. Pathogenicity was verified on H. arborescens and H. quercifolia by pressurised spray inoculation of the leaves with a suspension of 108 cfu/ml of each strain in PB in three replicates and with PB as negative control. Plants were covered for 48 hr with polythene sheet to support high humidity and facilitate infection. They were maintained in the greenhouse at 18/25°C (night/day). Leaf spots similar to those on the nursery plants developed on the inoculated leaves within four weeks. Koch's postulates were fulfilled by isolating from the leaf spots and by confirming the presence of identical gyrB sequences in re-isolated and inoculated strains. This is the first record of a bacterial leaf spot of Hydrangea in Belgium. A leaf spot disease of oak leaf hydrangea caused by a pathovar of Xanthomonas campestris was reported in the past in Georgia, USA (Uddin et al., 1996). This may have been a similar pathogen, alas the strain is not obtainable (R. Gitiatis, pers. comm.). The isolates were obtained from deciduous Hydrangea species. Infected plants that overwintered at the ILVO premises developed symptoms the following year but eventually the disease was no longer observed. Also, in the nurseries the disease has not been visible over the past few years. Stocks of Hydrangea plants are regularly renewed and summers have been hot and dry. The environmental conditions have certainly not been favourable for infection. The relevance of this pathogen is also underpinned by the presumed introduction in cuttings from the USA. It suggests that it is still present there after its first report in 1996. So, it may be re-introduced and re-emerge when conditions are more favourable. This article is based upon work from COST Action CA16107 EuroXanth, supported by COST (European Cooperation in Science and Technology).