First Report of Causing Leaf Spot on Oakleaf Hydrangea () in Tuscany (Italy).

Hydrangeas (Hydrangea L.) are popular ornamental plants used in urban landscapes and gardens worldwide for the beauty of their large flowers. In June 2022, dark brown/purple and irregular water-soaked spots coalescing into large areas of necrosis were observed on the leaves of potted Hydrangea quercifolia Bartr. plants growing in two ornamentals nurseries in Pistoia, Tuscany, Italy. Isolations, using two symptomatic plants/nursery, were performed by excising small portions of leaf tissue from the margin of the lesions, and macerating them in 100 μl of sterile distilled water (SDW). After 15 min of incubation, a loopful of the resulting suspension was streaked on yeast extract-dextrose-CaCO3 agar (YDCA) amended with 60 mg L-1 cycloheximide. Mucoid, convex and yellow colonies appeared on YDCA after incubation at 28°C for 48h. After colony purification on yeast extract-nutrient-agar (YNA), two isolates from each nursery were subject to amplification and sequence analysis of the 16S rRNA using universal primers FD1/RD1, for genus identification (Vauterin et al. 2000; Weisburg et al. 1991). All 16S rRNA sequences (OP441051) were identical and BLASTn searches indicated that the isolates belong to the genus Xanthomonas [99.9% nucleotide identity with X. hydrangeae strain LMG 31885 (LR990741.1) and 99.8% with strain LMG 31884T (NR_181958.1)]. For classification at species level, fragments of the housekeeping genes gyrB, rpoD, dnaK, and fyuA, were amplified according to Young et al. (2008). Both strands were sequenced and the consensus sequences aligned using MUSCLE as implemented in MEGA X (Kumar et al. 2018). Homologous sequences were once again identical between the isolates. A neighbor joining phylogenetic analysis of the concatenated fragments, was carried out, using the Tuscan isolate HyQ-Tu1, the type/pathotype strains of the seven pathovars of X.hortorum, proposed by Morinière et al. (2020), the four X.hydrangeae strains characterized by Dia et al. (2021) and the type strain of X.populi as the outgroup. The analysis indicated that HyQ-Tu1 isolate clusters within the X. hydrangeae branch of the recently described X. hortorum - X. hydrangeae species complex (Morinière et al. 2020; Dia et al. 2021; 2022). In agreement with this result, isolates tested positive to the LAMP assay specific for members of the complex's clade C (X. hydrangeae) (Dia et al. 2022). Based on molecular evidence, the isolates were identified as X. hydrangeae (Dia et al. 2021; Oren and Garrity, 2022). Three healthy H. quercifolia potted plants were inoculated by rubbing a 10 µl droplet of a bacterial suspension of X. hydrangeae HyQ-Tu1 adjusted to an OD600 of 0.3 (approx. 108 CFU/ml) in SDW on the adaxial surface of two leaves per plant. Two control leaves/plant were inoculated with SDW. Each inoculated leaf was enclosed for 24h in a polyethylene bag and all plants were maintained in a greenhouse at 28°C. Nine days post inoculation (DPI), leaf spots similar to those observed on naturally infected plants started to become evident on the bacteria-inoculated leaves while control leaves remained asymptomatic throughout the trial (21 DPI). Koch's postulates were fulfilled by re-isolating the bacterium from the symptomatic tissues, obtaining a positive amplification with the clade C-specific LAMP assay (Dia et al. 2022), and confirming that the gyrB sequence was 100% identical to that of X. hydrangeae HyQ-Tu1. Housekeeping gene sequences were submitted to GenBank (OP456006-9). Members of the X. hortorum - X. hydrangeae species complex have been reported to affect H. quercifolia in the USA (Uddin et al. 1996) and H. quercifolia and H. arborescens L. in Belgium (Cottyn et al. 2021). To the best of our knowledge, this is the first documentation of X. hydrangeae causing disease on H. quercifolia in Italy. Further work is required to verify the presence of the bacterium in other European countries and to assess the economic impact that it causes within and outside nurseries.