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.

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.

HomePlant DiseaseVol. 99, No. 5First Report of Bacterial Canker of Kiwifruit Caused by Pseudomonas syringae pv. actinidiae in Greece PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of Bacterial Canker of Kiwifruit Caused by Pseudomonas syringae pv. actinidiae in GreeceM. C. Holeva, P. E. Glynos, and C. D. KaraflaM. C. Holeva, P. E. Glynos, and C. D. KaraflaAffiliationsAuthors and Affiliations M. C. Holeva P. E. Glynos C. D. Karafla , Benaki Phytopathological Institute, Laboratory of Bacteriology, Kifissia, Greece. Published Online:29 May 2015https://doi.org/10.1094/PDIS-07-14-0738-PDNAboutSectionsSupplemental ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Bacterial canker of kiwifruit, caused by Pseudomonas syringae pv. actinidiae (Psa), was first described in Japan, South Korea, and China in the 1980s. Recently, the disease has been reported in New Zealand, Italy, Spain, France, Portugal, Slovenia, Switzerland, Turkey, Australia, and Chile, suggesting it can be considered as an international pandemic. The pathogen causes severe kiwifruit crop losses and has also been isolated from Actinidia arguta and A. kolomikta (2). In mid-March 2014, a sample of canes and leaders of A. deliciosa cv. Summer kiwi, showing red-rusty cankers and brown discoloration of the vascular tissues underneath the bark, was sent by a grower for examination to the Benaki Phytopathological Institute; the sample was originated from the area of Drosero Pellas (Macedonia, northern Greece). According to the sample information sheet, in a 1-ha orchard with 5-year-old plants, almost all plants exhibited symptoms. The bacterial isolates recovered from the cankers on nutrient agar with sucrose were gram negative, aerobic, levan positive, nonfluorescent on King's medium B, did not have a cytochrome c oxidase or an arginine dehydrolase activity, did not cause potato soft rot and did not hydrolyze starch or gelatin; they induced a hypersensitivity response on tobacco plants. These characteristics match those of Psa (3). Two representative isolates were selected for further testing, using two Psa strains as positive controls: CRA-FRU 8.43, provided by Dr. M. Scortichini (C.R.A.-Centro di Ricerca per la Frutticoltura, Roma, Italy) and CFBP 7286. DNA fragments of the expected size were amplified from genomic DNA of the two isolates using the Psa-specific primers: a) PsaF1/R2 (5) and b) KN-F/R and AvrDdpx-F/R (duplex PCR) (3). A multiplex PCR assay (1) further assigned the two strains to the 'European population' of Psa. In Box-PCR, the two isolates and the two Psa control strains produced similar banding patterns. The PsaF1/R2 amplicon (280-bp of the 16S-23S rDNA ITS region) of the two strains was sequenced (Beckman Coulter Genomics, UK) and found to be 100% identical to the strains CRA-FRU 8.43, CFBP 7286 (GenBank Accession No. AGNO01000048.1) and the pathotype strain ICMP 9617 (AY342165). Partial sequence (1,130-bp) of the 16S rDNA gene of the two isolates obtained with primers 63f/1389r (4) was 100% identical to CRA-FRU 8.43 and 99.73% identical to strains CFBP 7286 and ICMP 9617 (EU906856.1 and CM002753.1). Pathogenicity was confirmed by artificial inoculation of young, about 20 cm height, A. deliciosa cv. Hayward plants. The plants were inoculated by injecting a bacterial suspension (109 CFU/ml) into leaves with a hypodermic syringe or piercing leaves with a sterile needle through drops of the suspension placed on the leaf surfaces. First necrotic spots on leaves were observed 6 days after inoculation. No symptoms were observed on control plants similarly treated with sterile water. The bacteria isolated from necrotic spots were identified as Psa. To our knowledge this is the first report of bacterial canker of kiwifruit in Greece. A nationwide survey is underway to determine the extent of the affected area.References:(1) Balestra, G. M., et al. 2013. Plant Dis. 97:472. https://doi.org/10.1094/PDIS-06-12-0590-RE Link, ISI, Google Scholar(2) EPPO. 2014. PQR-EPPO database on quarantine pests (available online). http://www.eppo.int/DATABASES/pqr/pqr.htm. Google Scholar(3) Galleli, A., et al. 2011. J. Plant Pathol. 93:425. ISI, Google Scholar(4) Osborn, A. M., et al. 2000. Environ. Microbiol. 2:39. https://doi.org/10.1046/j.1462-2920.2000.00081.x Crossref, ISI, Google Scholar(5) Rees-George, J., et al. 2010. Plant Pathol. 59:453. https://doi.org/10.1111/j.1365-3059.2010.02259.x Crossref, ISI, Google ScholarDetailsFiguresLiterature CitedRelated Vol. 99, No. 5 May 2015SubscribeISSN:0191-2917e-ISSN:1943-7692 Metrics Article History Issue Date: 29 May 2015Published: 29 May 2015First Look: 13 Jan 2015Accepted: 30 Dec 2014 Pages: 723-723 Information© 2015 The American Phytopathological SocietyCited byThe Potential Global Climate Suitability of Kiwifruit Bacterial Canker Disease (Pseudomonas syringae pv. actinidiae (Psa)) Using Three Modelling Approaches: CLIMEX, Maxent and Multimodel Framework28 January 2022 | Climate, Vol. 10, No. 2Pseudomonas syringae pv. actinidiae (bacterial canker of kiwifruit)CABI Compendium, Vol. CABI CompendiumPolyphasic Analysis of Isolates from Kiwifruit Reveal New Genetic Lineages of Pseudomonas syringae pv. actinidifoliorum Look-Alike3 December 2021 | Agronomy, Vol. 11, No. 12Phage PPPL-1, A New Biological Agent to Control Bacterial Canker Caused by Pseudomonas syringae pv. actinidiae in Kiwifruit10 May 2021 | Antibiotics, Vol. 10, No. 5Pest survey card on Pseudomonas syringae pv.actinidiaeEFSA Supporting Publications, Vol. 17, No. 12Identification and Analysis of NBS-LRR Genes in Actinidia chinensis Genome13 October 2020 | Plants, Vol. 9, No. 10New insights about the complexity of Pseudomonas syringae pv. actinidiae across the worldActa Horticulturae, No. 1243Characterization of Pseudomonas syringae pv. actinidiae biovar 3 on kiwifruit in north-west Portugal24 July 2018 | Journal of Applied Microbiology, Vol. 125, No. 4Genomic Structural Variations Affecting Virulence During Clonal Expansion of Pseudomonas syringae pv. actinidiae Biovar 3 in Europe5 April 2018 | Frontiers in Microbiology, Vol. 9Microparticles containing gallic and ellagic acids for the biological control of bacterial diseases of kiwifruit plants27 April 2017 | Journal of Plant Diseases and Protection, Vol. 124, No. 6The Scientific, Economic, and Social Impacts of the New Zealand Outbreak of Bacterial Canker of Kiwifruit ( Pseudomonas syringae pv. actinidiae )Annual Review of Phytopathology, Vol. 55, No. 1Detection and characterization of Pseudomonas syringae pv. actinidifoliorum in kiwifruit in Spain22 November 2015 | Journal of Applied Microbiology, Vol. 119, No. 6Origin of the Outbreak in France of Pseudomonas syringae pv. actinidiae Biovar 3, the Causal Agent of Bacterial Canker of Kiwifruit, Revealed by a Multilocus Variable-Number Tandem-Repeat AnalysisApplied and Environmental Microbiology, Vol. 81, No. 19

In July 2005 and September 2006, samples of mature fruits of F1 hybrid watermelon (Citrullus lanatus) cv. Obla were received from the areas of Chryssoupoli (Macedonia, northern Greece) and Vagia (central Greece), respectively. Fruits had small, irregular, water-soaked lesions and brown cracks on their surface, brown discoloration and water-soaked areas in the rind underneath the lesions, and watery flesh rot. Disease incidence was reported as severe in both areas, according to the sample information sheets sent by local agronomists. Bacterial isolates recovered on nutrient agar (NA) from the affected fruits were Gram-negative, oxidase positive, non-fluorescent on King’s medium B, pathogenic to inoculated watermelon fruits and to seedlings of watermelon, melon, cucumber and pumpkin; isolates induced tobacco hypersensitivity and formed characteristic white colonies on nutrient agar. Based on these data, the isolates were identified as Acidovorax avenae subsp. citrulli (Aac). In May 2008, young grafted watermelon plants (F1 hybrid cv. Byblos) received from the area of Varda (Peloponnese, southern Greece) showed brown, angular, necrotic spots or larger lesions on leaves. This outbreak was reported by the agronomist in charge of the crop to have affected about 50% of a plot of 12 000 plants. The bacterial isolates recovered on NA from the affected plants showed the above properties and also growth at 41°C, no starch hydrolysis, oxidative glucose metabolism and utilization of d-galactose, d-glucose, l-arabinose, but not adonitol, arginine or sucrose, as sole carbon source. An immunofluorescence test with Aac-specific polyclonal antiserum (LOEWE, Germany) and sequencing (COGENICS, UK) of the products of PCR with two sets of Aac-specific primers, viz. BX-L1F/BX-R5F and BX-L1F/BX-S-R2R (Bahar et al., 2008), or the set 63f/1389r (Osborn et al., 2000) amplifying part of the 16S rDNA region, verified the isolates as Aac. In Rep-, Eric- or Box-PCR, the isolates from plants and Aac reference strains produced similar banding patterns. Koch’s postulates were fulfilled on seedlings and fruits of the above mentioned cucurbits with all isolates from plants. One such isolate was deposited in the Benaki Phytopathological Institute Culture Collection as BPIC2124. This is the first report confirming Aac naturally infecting watermelon plants and fruits in Greece. The authors wish to thank Dr S. Burdman for providing the Aac reference strains: W1, M1 (Bahar et al., 2008).