First report of causing bacterial wilt on sp. in Greece.

In March 2021, a sample of nine-month-old, non-grafted, diseased rose ( sp.) plants was sent by a grower to the Benaki Phytopathological Institute for examination. The plants exhibited symptoms of dieback with black necrosis of pruned shoots, brown discoloration of shoot and root vascular tissues, and whitish slime exudation on cutting wounds of the shoots. The symptoms resembled those caused by (Tjou-Tam-Sin et al. 2016). According to the sample's information sheet, the sample had been collected in a commercial greenhouse rose crop for cut flowers with a 10% disease incidence in the area of Troizinia-Methana (Regional Unit of Islands, Greece). Microscopic examination of symptomatic shoot and root vascular tissues revealed masses of bacterial cells streaming out of them. Sections of symptomatic tissues were suspended in water and in the resulting suspension, bacteria of the species complex (RSSC) were detected by an indirect immunofluorescence (IF) assay using polyclonal antibodies (Plant Research International, the Netherlands) and a qPCR assay (RS-I-F/RS-II-R primers, RSP-55T probe) (Vreeburg et al. 2016). Furthermore, colonies with typical characteristics of RSSC were isolated from vascular tissues of shoots and roots on non-selective (NA) and semi-selective (mSMSA) media (EPPO 2022), and their identification as RSSC was confirmed by the above-mentioned IF and qPCR assays. Also, the isolates were assigned to: i) biovar 3, based on their ability to metabolize three disaccharides (maltose, lactose, D(+) cellobiose) and three hexose alcohols (mannitol, sorbitol, dulcitol) producing acid (EU 2006) and ii) phylotype I, by multiplex conventional PCR (Opina et al. 1997; Fegan and Prior 2005). A representative isolate was selected for sequencing part of the genes: rDNA (1464bp), (729bp) and (795bp) with GenBank Accession Nos. OR102443, OR683617 and OR702781, respectively. Blast analysis of these sequences showed 100% identity with those of various RSSC strains (e.g. GenBank Ac. Nos. CP025741.1, CP021762.1, MF141029.1, respectively). The obtained sequence conforms with the characteristics of phylotype I based on the DNA barcoding tool (EPPO 2021) and is 100% identical to that of the Dutch strain PD7216 (MF141029.1) reported to be sequevar I-33 (Bergsma-Vlami et al. 2018). The pathogenicity of two isolates was tested by inoculating: i) tomato seedlings (cv. 'Belladona') at their stem between the cotyledons and the first true leaf (EU 2006) and b) rose plants (cv. 'Aqua' and 'Papa Meilland') at their shoot base (Tjou-Tam-Sin et al. 2016), with bacterial suspensions in water (10 cfu/ml). The inoculated plants were maintained at a day/night temperature about 28/20°C with tomato plants exhibiting leaf wilting (7-17 dpi) and rose plants exhibiting chlorosis and necrosis of leaves (17 dpi). The pathogen was re-isolated on mSMSA from both artificially infected plant species and identified by the IF assay described above, thus fulfilling Koch's postulates. This is the first diagnosis in Greece of: i) rose plants infected by a species and ii) a crop infected by phylotype I that corresponds to the species (EPPO 2022). Official phytosanitary measures imposed in the affected area include an annual survey of rose crops for the presence of this pathogen, aiming at an early detection and prevention of its spread in such a highly valued ornamental crop.