First report of Ralstonia pseudosolanacearum causing bacterial wilt on Rosa sp. in Greece.

In March 2021, a sample of nine-month-old, non-grafted, diseased rose (Rosa 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 Ralstonia pseudosolanacearum (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 R. solanacearum 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: 16S rDNA (1464bp), mutS (729bp) and egl (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 egl 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 (108 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 Ralstonia species and ii) a crop infected by R. solanacearum phylotype I that corresponds to the R. pseudosolanacearum 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.

First Report of the Occurrence of the Bacterial Spot Disease of Stone Fruits Caused by Xanthomonas arboricola pv. pruni on Almond in Greece.

In 2021, two samples of almond (Prunus dulcis (Mill) Webb) shoots with symptoms resembling those caused by Xanthomonas arboricola pv. pruni (Xap), were examined at the Benaki Phytopathological Institute. The first sample was collected in June from a 0.4-ha orchard of fifteen-year-old almond trees (cv. 'Texas') with 40% disease incidence, in the Regional Unit of Serres (Northern Greece). Leaves exhibited, mainly at their tip and margins, small, angular, necrotic spots with chlorotic halo, often coalesced into larger necrotic lesions which fell out leaving leaves with a 'shot-hole' like appearance. Fruits displayed dark brown, sunken, corky, gum oozing lesions and shoots developed dark brown, elongated, slightly sunken lesions. Bacterial streaming from the marginal areas of necrotic lesions was observed microscopically. On the lesions of fruits, leaves and shoots, Xap was detected by immunofluorescence assay (IF) using polyclonal antibodies (Plant Research International, the Netherlands) and two qPCR assays (Garita-Cambronero et al. 2017; Palacio-Bielsa et al. 2011). Eight Xanthomonas-like isolates obtained on the SP agar (Hayward 1960) and Nutrient agar (Schaad et al. 2001) culture media were Gram-negative, oxidase negative, strictly aerobic, sensitive to 0.1% w/v TTC, hydrolysing gelatin and Tween 80 but not starch, and also inducing hypersensitive response in tomato plants, as expected for Xap (Schaad et al. 2001). Isolates' identification was confirmed by the IF and the two qPCR assays cited above, as well as a conventional PCR (Pothier et al., 2011). Infiltration of a suspension (107 cfu/ml) of one isolate into five leaves of a two-year-old almond tree cv. 'Texas', and also into five detached leaves from the same tree (Randhawa and Civerolo 1985), caused necrotic spots on all inoculated leaves (10 inoculation sites/leaf), after a four day incubation period at 25oC under high humidity. The Xap reference strain NCPPB 3877 and sterile water were used as positive and negative controls, respectively. The pathogen was reisolated from necrotic spots of the inoculated leaves and identified by IF and two qPCR assays, as previously. The second sample was collected by a grower in September from a 3.7-ha orchard of five-year-old almond trees (cv. 'Tuono') exhibiting 50% disease incidence, in the Regional Unit of Fthiotida (Central Greece). Leaves and fruits showed symptoms similar to those described for the first sample, except that, lesions on fruits, which were at a stage of advanced mesocarp dehydration, were raised. Five Xap isolates were obtained from symptomatic leaves and fruits, and their pathogenicity on almond was confirmed, as in the first sample. Furthermore, sequences of PCR products using primers targeting the 16S-rDNA (Lane 1991;Lane et al., 1985), gyrB (Parkinson et al. 2007) and ftsX (Pothier et al. 2011) genes of two Xap isolates, one from fruit- and one from leaf-necrotic lesions of the first sample, were searched against the NCBI GenBank database, revealing that the obtained sequences of 16S-rRNA (OP412487; OP412488), gyrB (OP467593; OP467594) and ftsX (OP467595; OP467596) genes were 100% identical to the corresponding genomic regions of the Xap strains IVIA 2626.1 (CP076628.1) and CITA 33 (CP076701.1). This is the first report on the presence of Xap in Greece. As these Xap outbreaks have occurred in regions with extensive almond cultivation, a crop of great economic importance for Greece, measures for its eradication have already been advised.