The effectiveness of copper containing nanoparticles (Cu/CuO-NPs) against insecticide-resistant olive fruit flies (Bactrocera oleae) and their impact on the insect’s reproductive and endosymbiotic parameters were evaluated. The insecticidal activity of both nano and bulk copper [Cu(OH)2] was comparable or greater than that of the reference insecticide deltamethrin at recommended doses as revealed by feeding experiments. A significant synergistic effect between Cu-NPs or CuO-NPs and deltamethrin was observed in terms of adult mortality. Furthermore, the deltamethrin + Cu-NPs combination decreased the total number of offspring as compared with the untreated control. The above combination also significantly decreased the mean number of stings, pupae, female and total number offspring of the surviving female, compared to deltamethrin applied alone. The abundance of the Candidatus Erwinia dacicola- a B. oleae larvae bacterial gut endosymbiont- was adversely affected by bulk and nanosized copper. Concluding, Cu-NPs have a great potential to control insecticide-resistant B. oleae populations by reducing adult and larval survival and fecundity, and provide the means for reducing the environmental footprint of pesticides by minimizing their required doses. This study was co-financed by the European Regional Development Fund of the European Union and Greek national funds through the Rural Development Program (RDP/ΠΑΑ) 2014 – 2020, under the call "Cooperation for environmental projects, environmental practices and actions for climate change“ (project code: Μ16SΥΝ2-00354).

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.

BACKGROUND: The potential of copper nanoparticles (Cu-NPs) to be used as an alternative control strategy against olive fruit flies (Bactrocera oleae) with reduced sensitivity to the pyrethroid deltamethrin and the impact of both nanosized and bulk copper hydroxide (Cu(OH)(2)) on the insect's reproductive and endosymbiotic parameters were investigated. RESULTS: The application of nanosized and bulk copper applied by feeding resulted in significant levels of adult mortality, comparable to or surpassing those achieved with deltamethrin at recommended doses. Combinations of Cu-NPs or copper oxide nanoparticles (CuO-NPs) with deltamethrin significantly enhanced the insecticide's efficacy against B. oleae adults. When combined with deltamethrin, Cu-NPs significantly reduced the mean total number of offspring compared with the control, and the number of stings, pupae, female and total number of offspring compared with the insecticide alone. Both bulk and nanosized copper negatively affected the abundance of the endosymbiotic bacterium Candidatus Erwinia dacicola which is crucial for the survival of B. oleae larvae. CONCLUSION: The Cu-NPs can aid the control of B. oleae both by reducing larval survival and by enhancing deltamethrin performance in terms of toxicity and reduced fecundity, providing an effective anti-resistance tool and minimizing the environmental footprint of synthetic pesticides by reducing the required doses for the control of the pest. (c) 2024 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

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.

Cucumber mosaic virus (CMV) is a destructive plant virus with worldwide distribution and the broadest host range of any known plant virus, as well as a model plant virus for understanding plant–virus interactions. Since the discovery of RNA interference (RNAi) as a major antiviral defense, RNAi-based technologies have been developed for plant protection against viral diseases. In plants and animals, a key trigger of RNAi is double-stranded RNA (dsRNA) processed by Dicer and Dicer-like (DCL) family proteins in small interfering RNAs (siRNAs). In the present study, dsRNAs for coat protein (CP) and 2b genes of CMV were produced in vitro and in vivo and applied onto tobacco plants representing a systemic solanaceous host as well as on a local host plant Chenopodium quinoa. Both dsRNA treatments protected plants from local and systemic infection with CMV, but not against infection with unrelated viruses, confirming sequence specificity of antiviral RNAi. Antiviral RNAi was effective when dsRNAs were applied simultaneously with or four days prior to CMV inoculation, but not four days post inoculation. In vivo-produced dsRNAs were more effective than the in vitro-produced; in treatments with in vivo dsRNAs, dsRNA-CP was more effective than dsRNA-2b, while the effects were opposite with in vitro dsRNAs. Illumina sequencing of small RNAs from in vivo dsRNA-CP treated and non-treated tobacco plants revealed that interference with CMV infection in systemic leaves coincides with strongly reduced accumulation of virus-derived 21- and 22-nucleotide (nt) siRNAs, likely generated by tobacco DCL4 and DCL2, respectively. While the 21-nt class of viral siRNAs was predominant in non-treated plants, 21-nt and 22-nt classes accumulated at almost equal (but low) levels in dsRNA treated plants, suggesting that dsRNA treatment may boost DCL2 activity. Taken together, our findings confirm the efficacy of topical application of dsRNA for plant protection against viruses and shed more light on the mechanism of antiviral RNAi.

Tomato yellow leaf curl virus (TYLCV), a whitefly-transmitted single-stranded DNA (ssDNA) virus, causes the most important viral disease of tomato worldwide. TYLCV-mediated disease is mainly controlled via extensive insecticide sprays aiming at the whitefly vector. RNA-based vaccination was proven to be a non-transgenic approach leading to efficient plant virus control. In this work, double-stranded RNA (dsRNA) molecules deriving from sequences of the C4 and V2 genes of TYLCV-Mild were produced in vitro and topically applied onto tomato plants along with the virus (via agroinfiltration). DsC4 and dsV2 application reduced disease incidence to 23 and 46 %, respectively, while TYLCV positive control reached 64 %. Bioinformatics analysis of the virus-specific small interfering RNAs (vsiRNAs) from TYLCV-infected tomato revealed 'hot' and 'cold' spots in the TYLCV-Mild genome. Interestingly, the viral C-strand had twofold siRNA reads when compared to that of the V-strand. Overall, vsiRNAs of negative and positive polarity were almost equal (53.5 vs. 46.6 %); vsiRNAs of negative polarity prevailed at the V-strand. Stem-loop RT-PCR validated the presence of six vsiRNAs (hot or cold spots) in TYLCV-Mild-infected and dsRNA-treated tomato. The exogenously applied dsRNA was found to rapidly move systemically in tomato and was detected for 54 days post treatment (dpt). The applied dsRNA molecules were successfully processed by the Dicer-like proteins (DCLs) in tomato since small interfering RNAs (siRNAs) deriving from the dsRNA were detected for at least 54 dpt. This consists the first report of dsRNA-based vaccination applied against a monopartite geminivirus.

Bacteria in the genus Xanthomonas infect a wide range of crops and wild plants, with most species responsible for plant diseases that have a global economic and environmental impact on the seed, plant, and food trade. Infections by Xanthomonas spp. cause a wide variety of non-specific symptoms, making their identification difficult. The coexistence of phylogenetically close strains, but drastically different in their phenotype, poses an added challenge to diagnosis. Data on future climate change scenarios predict an increase in the severity of epidemics and a geographical expansion of pathogens, increasing pressure on plant health services. In this context, the effectiveness of integrated disease management strategies strongly depends on the availability of rapid, sensitive, and specific diagnostic methods. The accumulation of genomic information in recent years has facilitated the identification of new DNA markers, a cornerstone for the development of more sensitive and specific methods. Nevertheless, the challenges that the taxonomic complexity of this genus represents in terms of diagnosis together with the fact that within the same bacterial species, groups of strains may interact with distinct host species demonstrate that there is still a long way to go. In this review, we describe and discuss the current molecular-based methods for the diagnosis and detection of regulated Xanthomonas, taxonomic and diversity studies in Xanthomonas and genomic approaches for molecular diagnosis.

Samples of pear shoots, blossoms and leaves showing typical fire blight symptoms were collected from diseased pear trees during spring and summer over a period of 3 years from orchards situated in the Mitidja region, a main pear producing area in Northern Algeria. From all collected plant samples, thirty-seven putative Erwinia amylovora strains were isolated and identified by biochemical, serological and molecular tests. Molecular typing of these strains was performed using six variable number of tandem repeats sequences (VNTRs). Minimal spanning trees showed that four different haplotypes were present within the bacterial population analyzed. The efficacy of twenty potential bacterial antagonists was evaluated in vitro against two E. amylovora pathogenic strains. Four antagonists (Bacillus amyloliquefaciens, Bacillus methylotrophicus and two Pseudomonas brassicaceaurum) were selected to perform a biocontrol assay on immature pear fruits. Results showed that the two Pseudomonas strains were the most effective for preventive treatment on pear fruits, leading to a necrosis reduction of up to 90%. No curative effect was observed. The four selected antagonists were characterized for features linked to biocontrol activity, such as biofilm, siderophores and biosurfactant-production, motility, and effect of environmental conditions on bacterial growth.

Erwinia uzenensis is a plant-pathogenic bacterium, recently described in Japan, which infects pear trees, causing the 'bacterial black shoot disease of European pear' (BBSDP). Like other Erwinia pear pathogens, E. uzenensis causes damp, black lesions on young shoots resembling those of E. amylovora, but not blossom blight, fruitlet blight or wilting of the shoot tip. The distribution of E. uzenensis seems restricted to the country where it was reported up to now, but it may spread to other countries and affect new hosts, as is the current situation with E. piriflorinigrans and E. pyrifoliae. Fast and accurate detection systems for this new pathogen are needed to study its biology and to identify it on pear or other hosts. We report here the development of a specific and sensitive detection protocol based on a real-time PCR with a TaqMan probe for E. uzenensis, and its evaluation. In sensitivity assays, the detection threshold of this protocol was 101 cfu ml-1 on pure bacterial cultures and 102-103 cfu ml-1 on spiked plant material. The specificity of the protocol was evaluated against E. uzenensis and 46 strains of pear-associated Erwinia species different to E. uzenensis. No cross-reaction with the non-target bacterial species or the loss of sensitivity were observed. This specific and sensitive diagnostic tool may reveal a wider distribution and host range of E. uzenensis initially considered restricted to a region and will expand our knowledge of the life cycle and environmental preferences of this pathogen.

Due to the rapidly growing global population, food production and security is the major challenge of agriculture. Plant viruses are obligate parasites that in some instances could cause up to 100% losses in a crop (e.g. maize streak disease). Although difficult to accurately determine the global economic impact that plant viruses have on agriculture, it is estimated that US$60 billion loss in crop yields worldwide each year is due to plant viral diseases. RNA silencing (RNA interference, RNAi) is a conserved endogenous pathway of all higher eukaryotes, which controls gene expression. RNAi is induced by double-stranded RNA (dsRNA) and allows the cell to recognize aberrant genetic material in a highly sequence-specific manner ultimately leading to its degradation, thus protecting the cell from subcellular pathogens, such as viruses and transposons. DsRNA-mediated resistance has been exploited in transgenic plants to convey resistance to viruses and against insects, vectors of plant viruses, via host induced gene silencing (HIGS). A non-transgenic approach employing RNAi has been used where enzymatically synthesized specific dsRNA molecules, when applied directly onto plant tissue, induce resistance to the cognate virus; as a result dsRNA molecules could be efficacious antiviral agents for crop protection. Next generation sequencing and bioinformatics analyses have provided a plethora of information and useful tools for the design and study of dsRNA application. In this chapter, the different methods for dsRNA production, both in vitro and in vivo, the means of direct application of the dsRNA molecules onto plants and several examples of non-transgenic dsRNA-mediated resistance are presented.

Cucumber mosaic virus (CMV) causes great losses in Bhut Jolokia pepper (Capsicum chinense Jacq.) plantations in Assam, India. To investigate possible means to induce plant resistance against this virus, the crude extract of bacterially-expressed double-stranded (ds) RNA, derived from CMV-2b gene (dsRNA_CMV-2b), was exogenously applied along with CMV-G strain onto Bhut Jolokia plants. In this ‘RNA-vaccination’ bioassay, disease incidence, assessed by testing the plants at 21 days post inoculation by DAS-ELISA, ranged from 0 to 29% in case of dsRNA-treated plants, and from 55 to 92% when only CMV was applied. CMV-infected pepper plants became severely stunted, having dull light green foliage with leathery appearance, whereas plants receiving dsRNA_CMV-2b exhibited milder symptoms or remained healthy. The results obtained suggest that this non-transgenic approach has a considerable effect in protecting pepper against CMV.

The aim of this study was to characterise the performance of new molecular methods for the detection and identification of Pseudomonas syringae pv. actinidiae (Psa) and to provide validation data in comparison to the assays mentioned in official diagnostic protocols and being currently used. Eleven molecular tests for the Psa detection were compared in an inter-laboratory comparison where each laboratory had to analyse the same panel of samples consisting of thirteen Psa-spiked kiwifruit wood extracts. Laboratories had to perform also isolation from the wood extracts. Data from this interlaboratory test performance study (TPS) was statistically analysed to assess the performance of each method. In order to provide complete validation data, both for detection and identification, this TPS was supplemented by a further study of identification from pure culture of phylogenetically closely related Pseudomonas spp., Psa, and bacterial strains associated with kiwifruit. The results of both these studies showed that simplex-PCRs gave good results, whereas duplex-PCR and real-time PCR were the most reliable tools for detection and identification of Psa. Nested and multiplex-PCR gave false-positive results. The use of the most reliable detection test is suggested for routine analyses, but when Psa-free status needs to be accurately assessed, it is recommended that at least two detection tests are used. This work provides a wide comparison of the available diagnostic methods, giving new information for a possible revision of the official diagnostic protocols (e.g. European and Mediterranean Plant Protection Organization (EPPO) protocol PM7/120 for the detection of Psa).

Zucchini yellow mosaic virus (ZYMV) causes serious damage in a large number of cucurbits, and control measures are necessary. Transgenic cucurbits expressing parts of the ZYMV genome have been shown to be resistant to the cognate virus. A non-transgenic approach involving the exogenous application of double-stranded RNA (dsRNA) has also been shown to induce resistance in tobacco against Cucumber mosaic virus (CMV) and Tobacco mosaic virus (TMV). In the present study, dsRNA molecules derived from the helper component-proteinase (HC-Pro) and coat protein (CP) genes of the ZYMV_DE_2014 isolate were produced in vitro. On exogenous dsRNA application in cucumber, watermelon and squash plants, dsRNA HC-Pro conferred resistance of 82%, 50% and 18%, and dsRNA CP molecules of 70%, 43% and 16%, respectively. On deep sequencing analysis of ZYMV-infected watermelon, hot-spot regions for viral small interfering RNAs (vsiRNAs) in the genome of ZYMV were identified. Stem-loop reverse transcription-polymerase chain reaction (RT-PCR) detection of selected 21-nucleotide-long vsiRNAs in plants that received only dsRNA molecules suggested that the dsRNAs exogenously applied onto plants were successfully diced, thus initiating RNA silencing. dsRNA molecules were found to be progressively degraded in planta, and strongly detected by semi-quantitative RT-PCR for at least 9 days after exogenous application. Moreover, dsRNA molecules were detected in systemic tissue of watermelon and squash, showing that dsRNA is transported long distances in these plants.

Double-stranded RNA (dsRNA) is an inducer molecule of the RNA silencing (RNA interference, RNAi) pathway that is present in all higher eukaryotes and controls gene expression at the posttranscriptional level. This mechanism allows the cell to recognize aberrant genetic material in a highly sequence specific manner. This ultimately leads to degradation of the homologous target sequence, rendering the plant cell resistant to subcellular pathogens. Consequently, dsRNA-mediated resistance has been exploited in transgenic plants to convey resistance against viruses. In addition, it has been shown that enzymatically synthesized specific dsRNA molecules can be applied directly onto plant tissue to induce resistance against the cognate virus. This strongly implies that dsRNA molecules are applicable as efficacious agents in crop protection, which will fuel the demand for cost-effective dsRNA production methods. In this chapter, the different methods for dsRNA production-both in vitro and in vivo-are described in detail.

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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

Efficient control of Xanthomonas axonopodis pv. dieffenbachiae, the causal agent of anthurium bacterial blight, requires sensitive and reliable diagnostic tools. The European standard EN ISO 16140:2003 has been followed to compare a nested PCR assay (N-PCR) to a reference method (isolation and serological identification of bacterial colonies) and to other alternative serological detection methods. The evaluation was performed in two steps: a comparative study and a collaborative study involving 15 European laboratories. Although inclusivity was maximal (100%) for all methods, a maximal exclusivity was obtained only with N-PCR followed by an enzymatic restriction digestion of the amplicons. Exclusivity indices of 906, 887 and 472% were found for indirect ELISA, immunofluorescence and double antibody sandwich ELISA, respectively. An exclusivity of 925% was obtained with the reference method, further increased to 100% if pathogenicity tests were performed as a supplemental assay. The best level of sensitivity (relative detection level) was obtained with the reference method followed by the N-PCR assay. The N-PCR performance in terms of relative accuracy, accordance and concordance was very similar to that of the reference method. Moreover, N-PCR had undeniable advantages compared to the reference method (less labour-intensive and less time-consuming). In addition, post-test probabilities of infection were calculated to select the most appropriate detection scheme related to the prevalence of the pathogen. The N-PCR assay has since been included in a revised version of the EPPO detection protocol.

EFSA guidance regarding pest risk assessment previously identified limitations within a generic qualitative risk assessment scheme published to support EU plant health decision making. In seeking to develop methods to overcome the limitations, we report results of a multiphase project where five test risk assessment methods were applied to Anoplophora glabripennis, Candidatus Phytoplasma pyri, Guignardia citricarpa, Meloidogyne chitwoodi, and Xanthomonas citri strains causing citrus bacterial canker. Teams of pest risk assessors applied the methods to the pests and compared methods in relation to EFSA PLH needs. The most promising method was then revised to incorporate desirable features from the other methods and tested on five more case study pests; Acidovorax citrulli, Ca.P. mali, Ca. P. prunorum, M. Fallax and Mycosphaerella dearnessii. In addition the risk assessment method that emerged from the EU funded project PRATIQUE was adapted to better suit EFSA needs and also tested on the second set of pests. Comprehensive datasheets on each pest were compiled to support the risk assessments. Datasheets included information that emerged from individual pest questionnaires distributed to the National Plant Protection Organisations of each EU Member State. Short term experimental studies were conducted to reduce uncertainty regarding survival and infectivity of the root-knot nematodes in the absence of host plants. Small scale experiments also clarified the role of vectors in the spread of Ca. Phytoplasma prunorum, proving that psyllids acquired the pathogen from wild Prunus sp. and transmitted it to orchards. Results from both sets of studies better informed the respective risk assessments and reduced some uncertainties although significant uncertainties still remain elsewhere within assessments. A novel method to determine an index of individual risk components, e.g. likelihood of entry, was developed. The performance of risk reduction options was evaluated by repeating assessments considering scenarios with and without risk reduction options in place. (Resume d'auteur)