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In 2017, Xylella fastidiosa, a quarantine plant-pathogenic bacterium in Europe, was detected in almond trees associated with leaf scorch symptoms in Alicante, a Mediterranean area in southeastern mainland Spain. The bacterium was detected by serological and molecular techniques, isolated in axenic culture from diseased almond trees, and identified as X. fastidiosa subsp. multiplex sequence type (ST) 6. Inoculation experiments on almond plants in greenhouse trials with a characterized strain of X. fastidiosa subsp. multiplex ST6 isolated in the outbreak area have proved that it was able to multiply and systemically colonize inoculated plants. Disease symptoms characteristic of leaf scorch like those observed in the field were observed in the inoculated almond trees after 1 year. Furthermore, the pathogen was reisolated and identified by molecular tests. With the fulfillment of Koch’s postulates, we have demonstrated that X. fastidiosa is the causal agent of the almond leaf scorch disease in the Alicante outbreak.

Xylella fastidiosa (Xf) is a vascular plant pathogenic bacterium native to the Americas that is causing significant epidemics and economic losses in olive and almonds in Europe, where it is a quarantine pathogen. Since its first detection in 2013 in Italy, mandatory surveys across Europe revealed the presence of the bacterium also in France, Spain, and Portugal. Combining ONT and Illumina sequencing data, we assembled high-quality complete genomes of seven Xf subsp. fastidiosa strains isolated from different plants in Spain, the USA, and Mexico. Comparative genomic analyses discovered differences in plasmid content among strains, including plasmids that had been overlooked previously when using Illumina sequencing platform alone. Interestingly, in strain CFBP8073, intercepted in France from plants imported from Mexico, three plasmids were identified, including two (plasmid pXF-P1.CFBP8073 and pXF-P2.CFBP8073) not previously described in X. fastidiosa, and one (pXF5823.CFBP8073) almost identical to a plasmid described in a Xf strain from citrus. Plasmids found in the Spanish strains here were similar to those described previously in other strains from the same subspecies and ST1 isolated in the Balearic Islands and the USA. The genome resources from this work will assist in further studies on the role of plasmids in the epidemiology, ecology, and evolution of this plant pathogen., This research was funded by Project BeXyl (Beyond Xylella, Integrated Management Strategies for Mitigating Xylella fastidiosa impact in Europe; Grant ID No 101060593) from European Union’s Horizon Action ‘Food, Bioeconomy Natural Resources, Agriculture and Environment’ Programme; Project E-RTA2017-00004-C06-02 (Desarrollo de estrategias de erradicación, contención y control de X. fastidiosa enEspaña) from ‘Programa Estatal de I+D+I Orientada a los Retos de la Sociedad of the Spanish Government AEI-INIA Spain’ and The Spanish Olive Oil Interprofesional; and the Thematic Interdisciplinary Platform on X. fastidiosa from CSIC (PTI Sol-Xyl). Financial support from the Spanish Ministry of Agriculture and Fisheries to the Institute for Sustainable Agriculture is also acknowledged. L.D.F. was a recipient of a research fellowship awarded by the Organization for Economic Co-operation and Development (OECD), Co-operative Research Programme (CRP), during the writing of this manuscript. M. P. Velasco Amo is recipient of a contract financed by the Intramural Project 178 201840E111 from CSIC.

Huanglongbing (HLB) is the most devastating citrus disease and is associated with three bacterial species of the genus &lsquo, Candidatus Liberibacter&rsquo, transmitted by insect vectors. The early detection of HLB is based on PCR methods, and it is one of the cornerstones for preventing incursion into disease-free countries. However, the detection of phytopathogenic bacteria with PCR-based methods is problematic in surveys that include a variety of samples of different origins. Here, we first report the proportion of amplifications obtained by two standardized real-time PCR methods for the diagnosis of HLB in various environmental samples that include plants, psyllid vectors, and parasitic wasps of the psyllids. The results of 4915 samples showed that 9.3% of them were amplified by the first rapid screening test and only 0.3% by the more specific tests. Most of the amplifications were associated with parasitic wasps. We designed the primers external to the target regions of both real-time PCR protocols to determine if amplifications belonged to one of three &lsquo, Ca. Liberibacter&rsquo, species associated with HLB. The bioinformatic analysis of the sequences obtained with these primers revealed that all these amplifications came from the presence of other prokaryotic organisms in the samples. The primers developed in this study overcome the problem of undesired amplification in environmental samples. Thus, they could be used in future survey protocols to prevent the eradication of negative trees and the generation of unjustified alarms.

Control of bacterial plant diseases is a major concern, as they affect economically important species and spread easily, such as the case of fire blight of rosaceous caused by Erwinia amylovora. In the search for alternatives to the use of agrochemicals and antibiotics, this work presents a screening of natural bacterial antagonists of this relevant and devastating phytopathogen. We recovered bacterial isolates from different plant tissues and geographical origins and then selected those with the strongest ability to reduce fire blight symptoms ex vivo and remarkable in vitro antagonistic activity against E. amylovora. None of them elicited a hypersensitivity reaction in tobacco leaves, most produced several hydrolytic enzymes and presented other biocontrol and/or plant growth-promoting activities, such as siderophore production and phosphate solubilization. These isolates, considered as biocontrol candidates, were identified by 16S rRNA sequencing as Pseudomonas rhizosphaerae, Curtobacterium flaccumfaciens, Enterobacter cancerogenus, Pseudomonas azotoformans, Rosenbergiella epipactidis and Serratia plymuthica. This is the first time that the last five bacterial species are reported to have biocontrol potential against E. amylovora.

In the outbreak of Xylella fastidiosa in the province of Alicante (mainland Spain), all isolates characterized so far belong to subsp. multiplex and ST6, and the main host is Prunus dulcis. Two different strains (IVIA 5901 and ESVL), isolated from almond trees in two municipalities of the Demarcated Area, were used to carry out pathogenicity tests on different plant species. The Italian strain De Donno (subsp. pauca, ST53) and the Spanish strain IVIA 5770 (subsp. fastidiosa, ST1) were also included. Five cultivars of Prunus dulcis, eight of Olea europaea, four of Vitis vinifera, Citrus limon, Citrus sinensis, Citrus reticulata, Diospyros kaki, Eriobotrya japonica, and the forest species Quercus suber and Q. ilex were challenged. A total of 35 plants for each species or cultivar were inoculated. At 1, 3, 6, 12 and 18 months post-inoculation (mpi) plants were sampled and analyzed by real-time PCR, to assess bacterial movement over time. The results show that, in general, no clear pattern of movement was found in the different plant species. Interestingly, in olive and grape, regardless the cultivar, the subsp. multiplex ST6 Alicante isolates were hardly detected from 12 mpi onwards, i.e. the systemic movement was very slow. In fact, they were only detected in less than 4% of the infected plants and with very late Cq values. In citrus plants, detection of these isolates failed since 3 mpi, as occurred in kaki and loquat trees. As for Quercus species, the detection rate was higher in Q. suber than in Q. ilex. Only in almond and cork oak trees, leaf scorch symptoms could be correlated with the presence of X. fastidiosa subsp. multiplex. Moreover, in these cases the pathogen was re-isolated. The timing and the level of detection for all the strains throughout the trial obtained so far are presented.

In September 2019, symptoms resembling those of bacterial leaf blight were observed on carrot plants (

Vegetative disorders similar to those associated with the presence of ‘Candidatus Liberibacter solanacearum’ (CaLsol) were observed in carrot plants in Kairouan, Tunisia from 2014 to 2016. Symptoms including leaf curling, yellowing, bronze and purplish discoloration, stunting of plants and roots, and proliferation of secondary roots, affected 20 to 40% of the carrots in some plots. In order to determine if these symptoms were associated with the presence of CaLsol, and/or ‘Ca. Phytoplasma spp.’ and/or Spiroplasma citri, real-time PCR analyses were conducted using specific primers for these pathogens. CaLsol was detected for the first time in Tunisia and for the first time its haplotypes D and E were detected co-infecting a carrot plant. Furthermore, three samples of carrot seed produced in Kairouan tested positive for the haplotype D, showing a high percentage (35 to 63%) of viable bacterial cells after treatment with propidium monoazide. However, all the tests were negative for ‘Ca. Phytoplasma’ spp. as well as for S. citri. The results highlight that several CaLsol haplotypes are emerging carrot pathogens in new areas.

Pathogen introductions have led to numerous disease outbreaks in naive regions of the globe. The plant pathogen Xylella fastidiosa has been associated with various recent epidemics in Europe affecting agricultural crops, such as almond, grapevine, and olive, but also endemic species occurring in natural forest landscapes and ornamental plants. We compared whole-genome sequences of X. fastidiosa subspecies multiplex from America and strains associated with recent outbreaks in southern Europe to infer their likely origins and paths of introduction within and between the two continents. Phylogenetic analyses indicated multiple introductions of X. fastidiosa subspecies multiplex into Italy, Spain, and France, most of which emerged from a clade with limited genetic diversity with a likely origin in California, USA. The limited genetic diversity observed in X. fastidiosa subspecies multiplex strains originating from California is likely due to the clade itself being an introduction from X. fastidiosa subspecies multiplex populations in the southeastern United States, where this subspecies is most likely endemic. Despite the genetic diversity found in some areas in Europe, there was no clear evidence of recombination occurring among introduced X. fastidiosa strains in Europe. Sequence type taxonomy, based on multilocus sequence typing (MLST), was shown, at least in one case, to not lead to monophyletic clades of this pathogen; whole-genome sequence data were more informative in resolving the history of introductions than MLST data. Although additional data are necessary to carefully tease out the paths of these recent dispersal events, our results indicate that whole-genome sequence data should be considered when developing management strategies for X. fastidiosa outbreaks., This work was funded by the following awards: XF-ACTORS (Xylella fastidiosa Active Containment Through a Multidisciplinary-Oriented Research Strategy, grant 727987 from the European Union’s Horizon 2020 Framework Research Programme), E-RTA2017-00004-C02 (Desarrollo de estrategias de erradicación, contención y control de X. fastidiosa en España from “Programa Estatal de I+D+I Orientada a los Retos de la Sociedad of the Spanish Government” and FEDER), the Spanish Olive Oil Interprofessional, and the California Department of Food and Agriculture Pierce’ Disease Research Program.

Fire blight is a devastating plant disease caused by the bacterium Erwinia amylovora, and its control is frequently based on the use of copper-based compounds whose mechanisms of action are not well known. Consequently, in this article, we investigate the response of E. amylovora to copper shock by a whole-genome microarray approach. Transcriptional analyses showed that, in the presence of copper, 23 genes were increased in expression; these genes were classified mainly into the transport and stress functional categories. Among them, the copA gene was strongly induced and regulated in a finely tuned manner by copper. Mutation of copA, soxS, arcB, yjcE, ygcF, yhhQ, galF and EAM_3469 genes revealed that tolerance to copper in E. amylovora can be achieved by complex physiological mechanisms, including: (i) the control of copper homeostasis through, at least, the extrusion of Cu(I) by a P-type ATPase efflux pump CopA; and (ii) the overcoming of copper toxicity caused by oxidative stress by the expression of several reactive oxygen species (ROS)-related genes, including the two major transcriptional factors SoxS and ArcB. Furthermore, complementation analyses demonstrated the important role of copA for copper tolerance in E. amylovora, not only in vitro, but also in inoculated pear shoots.

An outbreak of Xylella fastidiosa subsp. multiplex sequence type ST6 was discovered in 2017 in mainland Spain affecting almond trees. Two cultured almond strains, “ESVL” and “IVIA5901,” were subjected to high throughput sequencing and the draft genomes assembled. Phylogenetic analysis conclusively indicated they belong to the subspecies multiplex, and pairwise comparisons of the chromosomal genomes showed an average nucleotide identity higher than 99%. Interestingly, the two strains differ for the presence of the plasmids pXF64-Hb_ESVL and pUCLA-ESVL detected only in the ESVL strain. The availability of these draft genomes contribute to extend the European genomic sequence dataset, a first step toward setting new research to elucidate the pathway of introduction and spread of the numerous strains of this subspecies so far detected in Europe., This work was funded by European Union’s Horizon 2020 Framework Research Programme Projects XF-ACTORS (Xylella fastidiosa Active Containment Through a Multidisciplinary-Oriented Research Strategy grant 727987) and MSCA-RISE-2016 CURE-XF (Capacity Building and Raising Awareness in Europe and in Third Countries to Cope with Xylella fastidiosa); COST Action CA16107 EuroXanth, supported by European Cooperation in Science and Technology; and Project Desarrollo de estrategias de erradicación, contención y control de en España: Diagnóstico, estructura genética y gama de huéspedes project E-RTA2017-00004-C06-02 from Programa Estatal de I+D+I Orientada a los Retos de la Sociedad of the Spanish Government. This work used the Vincent J. Coates Genomics Sequencing Laboratory at University of California, Berkeley, supported by NIH S10 OD018174 Instrumentation Grant.

Se ha construido un robot electrico asistido por control remoto para inspeccionar cultivos horticolas mediante teledeteccion, embarcando tres camaras reflex (dos modificadas para NIR y BNDVI), una termica, otra multiespectral (ocho bandas entre 550 y 850 nm) y otra hiperespectral (400-1000 nm). Las camaras estan colocadas mirando al suelo (plantas) y para controlar la iluminacion se utilizan cuatro focos halogenos en la zona de inspeccion que ha sido protegida de la luz exterior con una lona. Las imagenes se han georeferenciado utilizado un receptor GNSS (resolucion de 3 cm). Para controlar el avance del robot y sincronizar la adquisicion de imagenes, se ha desarrollado una placa electronica a medida utilizando un sensor inductivo acoplado a las ruedas del robot y un software a medida instalado en un computador industrial. Se han realizado inspecciones mensuales en dos parcelas experimentales de zanahorias en Villena (Espana) para detectar plantas asintomaticas infectadas por Candidatus Liberibacter solanacearum. Se han creado mapas del campo utilizando indices vegetativos a partir de los datos espectrales con resoluciones entre 1 y 2,5 mm por pixel. Durante el ultimo ensayo, se etiquetaron 100 plantas que se recolectaron y analizaron utilizando PCR en tiempo real para comparar los resultados con el analisis de las imagenes.

We report the complete annotated genome sequence of the plant-pathogenic bacterium Xylella fastidiosa subsp. fastidiosa strain IVIA5235.

We report the complete annotated genome sequence of the plant-pathogenic bacterium Xylella fastidiosa subsp. fastidiosa strain IVIA5235., We report the complete annotated genome sequence of the plant-pathogenic bacterium Xylella fastidiosa subsp. fastidiosa strain IVIA5235. This strain was recovered from a cherry tree in Mallorca, Spain.

International audience; Three isolates obtained from symptomatic nectarine trees (Prunus persica var. nectarina) cultivated in Murcia, Spain, which showed yellow and mucoid colonies similar to Xanthomonas arboricola pv. pruni, were negative after serological and real-time PCR analyses for this pathogen. For that reason, these isolates were characterized following a polyphasic approach that included both phenotypic and genomic methods. By sequence analysis of the 16S rRNA gene, these novel strains were identified as members of the genus Xanthomonas, and by multilocus sequence analysis (MLSA) they were clustered together in a distinct group that showed similarity values below 95 % with the rest of the species of this genus. Whole-genome comparisons of the average nucleotide identity (ANI) of genomes of the strains showed less than 91 % average nucleotide identity with all other species of the genus Xanthomonas. Additionally, phenotypic characterization based on API 20 NE, API 50 CH and BIOLOG tests differentiated the strains from the species of the genus Xanthomonas described previously. Moreover, the three strains were confirmed to be pathogenic on peach (Prunus persica), causing necrotic lesions on leaves. On the basis of these results, the novel strains represent a novel species of the genus Xanthomonas, for which the name Xanthomonas prunicola is proposed. The type strain is CFBP 8353 (=CECT 9404=IVIA 3287.1).

The characterization and intraspecific diversity of a collection of 45 Ralstonia solanacearum strains isolated in Spain from different sources and geographical origins is reported. To test the influence of the site and the host on strain diversity, phenotypic and genotypic analysis were performed by a polyphasic approach. Biochemical and metabolic profiles were compared. Serological relationship was evaluated by Indirect-ELISA using polyclonal and monoclonal antibodies. For genotypic analysis, hrpB and egl DNA sequence analysis, repetitive sequences (rep-PCR), amplified fragment length polymorphism (AFLP) profiles and macrorestriction with XbaI followed by pulsed field gel electrophoresis (PFGE) were performed. The biochemical and metabolic characterization, serological tests, rep-PCR typing and phylogenetic analysis showed that all analysed strains belonged to phylotype II sequevar 1 and shared homogeneous profiles. However, interesting differences among strains were found by AFLP and macrorestriction with XbaI followed by PFGE techniques, some profiles being related to the geographical origin of the strains. Diversity results obtained offer new insights into the biogeography of this quarantine organism and its possible sources and reservoirs in Spain and Mediterranean countries.

Xylella fastidiosa, a quarantine organism in the European Union (EU), causes diseases in a wide variety of plants such as almond, cherry, grape, citrus, elm, olive, and coffee trees and many ornamental plants. Since the detection of the bacterium in Italy (2013), where it is associated to a severe epidemic on olive trees, the pathogen has also been detected in France (2015) and Germany (2016) (EPPO 2016)., The work was supported by funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 635646 POnTE (Pest Organisms Threatening Europe) and No 727987 XF-ACTORS (Xylella Fastidiosa Active Containment Through a multidisciplinary-Oriented Research Strategy), and the Spanish Ministry of Agriculture and Fisheries, Food and Enviroment (MAPAMA).

[EN] Xanthomonas arboricola pv. pruni is a quarantine pathogen and the causal agent of the bacterial spot disease of stone fruits and almond, a major threat to Prunus species. Rapid and specific detection methods are essential to improve disease management, and therefore a prototype of a lateral flow immunoassay (LFIA) was designed for the detection of X. arboricola pv. pruni in symptomatic field samples. It was developed by producing polyclonal antibodies which were then combined with carbon nanoparticles and assembled on nitrocellulose strips. The specificity of the LFIA was tested against 87 X. arboricola pv. pruni strains from different countries worldwide, 47 strains of other Xanthomonas species and 14 strains representing other bacterial genera. All X. arboricola pv. pruni strains were detected and cross-reactions were observed only with four strains of X. arboricola pv. corylina, a hazelnut pathogen that does not share habitat with X. arboricola pv. pruni. The sensitivity of the LFIA was assessed with suspensions from pure cultures of three X. arboricola pv. pruni strains and with spiked leaf extracts prepared from four hosts inoculated with this pathogen (almond, apricot, Japanese plum and peach). The limit of detection observed with both pure cultures and spiked samples was 10(4) CFU ml(-1). To demonstrate the accuracy of the test, 205 samples naturally infected with X. arboricola pv. pruni and 113 samples collected from healthy plants of several different Prunus species were analyzed with the LFIA. Results were compared with those obtained by plate isolation and real time PCR and a high correlation was found among techniques. Therefore, we propose this LFIA as a screening tool that allows a rapid and reliable diagnosis of X. arboricola pv. pruni in symptomatic plants., The work was supported by the following: Instituto Nacional de Tecnologia Agraria y Alimentaria, Project RTA-2011-00140-C03-01 (http://www.inia.es), PLS MTG EMN MML; Instituto Nacional de Tecnologia Agraria y Alimentaria, FPI-INIA grant (http://www.inia.es), PLS; Generalitat Valenciana (Prometeo II 2014/040) (http://www.gva.es), PN RP AM; Ministerio de Economia y Competitividad (MINECO) (CTQ2013-45875R) (http://www.mineco.gob.es), PN RP AM; European Social Fund, PLS MTG EMN MML; and European Regional Development Fund, PLS MTG EMN MML.

Fire blight is a devastating plant disease caused by the bacterium Erwinia amylovora, and its control is frequently based on the use of copper‐based compounds whose mechanisms of action are not well known. Consequently, in this article, we investigate the response of E. amylovora to copper shock by a whole‐genome microarray approach. Transcriptional analyses showed that, in the presence of copper, 23 genes were increased in expression; these genes were classified mainly into the transport and stress functional categories. Among them, the copA gene was strongly induced and regulated in a finely tuned manner by copper. Mutation of copA, soxS, arcB, yjcE, ygcF, yhhQ, galF and EAM_3469 genes revealed that tolerance to copper in E. amylovora can be achieved by complex physiological mechanisms, including: (i) the control of copper homeostasis through, at least, the extrusion of Cu(I) by a P‐type ATPase efflux pump CopA; and (ii) the overcoming of copper toxicity caused by oxidative stress by the expression of several reactive oxygen species (ROS)‐related genes, including the two major transcriptional factors SoxS and ArcB. Furthermore, complementation analyses demonstrated the important role of copA for copper tolerance in E. amylovora, not only in vitro, but also in inoculated pear shoots.

The bacterium Erwinia amylovora causes fire blight, a serious and widespread disease of several pome fruit and ornamental plants. The use of suitable detection tools is essential for preventing its dissemination and, according to the protocol of the European and Mediterranean Plant Protection Organization, the isolation and further identification of E. amylovora is the only conclusive test of its presence. However, bacterial growth on solid media can be hampered when the pathogen is suffering stressful conditions in pome fruit or in other habitats. Since copper is an essential micronutrient that, in E. amylovora, also increases the exopolysaccharide production in rich-nutrient media, we have designed a non-selective differential medium containing 1.5 mM CuSO4 to improve the recovery of E. amylovora from plants under unfavorable conditions. In this new medium named Recovery Erwinia amylovora-Stressed Cells (RESC), its colonies were easily distinguished by a light yellow color and a high mucus production. The plating recovery of several E. amylovora strains in vitro and from naturally infected samples was significantly improved with respect to other media routinely employed, particularly when the pathogen was suffering stressful conditions. Thus, the recovery of stressed E. amylovora cells (after UV irradiation, nutrient deprivation, or the presence of copper ions in non-copper-complexing media) was significantly enhanced on RESC medium, and their culturability period extended. Therefore, RESC is a useful and valuable medium for the isolation of E. amylovora when adverse conditions in the natural environment are expected.

Monitoring the ability of bacterial plant pathogens to survive in insects is required for elucidating unknown aspects of their epidemiology and for designing appropriate control strategies. Erwinia amylovora is a plant pathogenic bacterium that causes fire blight, a devastating disease in apple and pear commercial orchards. Studies on fire blight spread by insects have mainly focused on pollinating agents, such as honeybees. However, the Mediterranean fruit fly (medfly) Ceratitis capitata (Diptera: Tephritidae), one of the most damaging fruit pests worldwide, is also common in pome fruit orchards. The main objective of the study was to investigate whether E. amylovora can survive and be transmitted by the medfly. Our experimental results show: i) E. amylovora can survive for at least 8 days inside the digestive tract of the medfly and until 28 days on its external surface, and ii) medflies are able to transmit the bacteria from inoculated apples to both detached shoots and pear plants, being the pathogen recovered from lesions in both cases. This is the first report on E. amylovora internalization and survival in/on C. capitata, as well as the experimental transmission of the fire blight pathogen by this insect. Our results suggest that medfly can act as a potential vector for E. amylovora, and expand our knowledge on the possible role of these and other insects in its life cycle.

Copper compounds, widely used to control plant-pathogenic bacteria, have traditionally been employed against fire blight, caused by Erwinia amylovora. However, recent studies have shown that some phytopathogenic bacteria enter into the viable-but-nonculturable (VBNC) state in the presence of copper. To determine whether copper kills E. amylovora or induces the VBNC state, a mineral medium without copper or supplemented with 0.005, 0.01, or 0.05 mM Cu2+ was inoculated with 107 CFU/ml of this bacterium and monitored over 9 months. Total and viable cell counts were determined by epifluorescence microscopy using the LIVE/DEAD kit and by flow cytometry with 5-cyano-2,3-ditolyl tetrazolium chloride and SYTO 13. Culturable cells were counted on King's B nonselective solid medium. Changes in the bacterial morphology in the presence of copper were observed by scanning electron microscopy. E. amylovora entered into the VBNC state at all three copper concentrations assayed, much faster when the copper concentration increased. The addition of different agents which complex copper allowed the resuscitation (restoration of culturability) of copper-induced VBNC cells. Finally, copper-induced VBNC cells were virulent only for the first 5 days, while resuscitated cells always regained their pathogenicity on immature fruits over 9 months. These results have shown, for the first time, the induction of the VBNC state in E. amylovora as a survival strategy against copper. Fire blight, caused by the bacterium Erwinia amylovora (Burrill) Winslow et al. (39) and reported in more than 40 countries around the world, is a very serious and destructive disease of pome fruits and many ornamental plants from the Rosaceae family (34). Copper compounds, widely utilized against fire blight from the beginning of the last century (35), are still employed in many countries, especially in the European Union, where antibiotic utilization is restricted (2). Their use is one of the most common methods for controlling bacterial plant diseases, but it has led many bacteria to develop different strategies against copper ions (31). Until now, very little information on the interaction between E. amylovora and Cu2+ ions has been available (4, 15, 41). Copper treatments have traditionally been considered as bactericides in agriculture (29, 34, 35), their effectiveness often being measured by the absence of bacterial growth on a solid medium (12, 13). However, recent studies have shown the induction of the viable-but-nonculturable (VBNC) state by copper in several plant-pathogenic bacteria, such as Agrobacterium tumefaciens (1), Xanthomonas campestris pv. campestris (16), and Ralstonia solanacearum (17). This state, in which cells progressively lose their culturability on nonselective solid medium but still remain viable, is considered to be a bacterial survival strategy under adverse environmental conditions (30). Therefore, the failure to produce a visible colony may not necessarily mean that the bacterial cell is dead. Furthermore, it has been reported that VBNC cells can maintain pathogenicity (17, 19). In this respect, it has been suggested that copper-induced VBNC cells of some phytopathogenic bacteria could be related to the persistent nature of infections in copper-treated fields (17). A similar situation could occur with E. amylovora, since fire blight remains difficult to control as a disease (26), which has been related to the ability of this bacterium to survive and spread in different ways (32). Moreover, some bacteria, under favorable environmental conditions, can revert from the VBNC state to a culturable one, in a process usually called resuscitation. This reversion is considered a confirmation that the VBNC state is a bacterial survival strategy (19, 28). The nature of the VBNC state, however, is still the topic of an intense debate in the literature, and some authors argue that this condition may be a physiological state prior to cell death (7, 23). In spite of previous work concerning the toxic effect of copper ions for E. amylovora (15), many questions on the survival of this bacterium in the presence of this metal remain unanswered. Thus, the aim of this work has been to determine whether copper kills or induces the VBNC state of E. amylovora cells and if such VBNC cells retain their pathogenicity. Furthermore, the possible reversion of this bacterium from the nonculturable state has been studied as well as whether resuscitated cells could regain their pathogenic potential

The role of irrigation water in disseminating Erwinia amylovora is not fully recognized, and the survival of the bacterium in natural water has not been carefully investigated. This risk has been underestimated, since it is generally considered that E. amylovora survives only for a short period in water and its isolation from natural water samples has not been reported. The main goal of this study has been to clarify whether E. amylovora could survive under nutrient starvation conditions usually found in aquatic environments and if it is nonculturable on solid media when recovered from sterile natural water. Infectivity of E. amylovora cells in water was evaluated by using immature pear fruits. Total and viable cell counts were monitored by the Live/Dead viability kit, and culturability by plate counts on King's B medium. E. amylovora was able to survive in water from different sources showing a long persistence in irrigation water and maintaining its infectivity for green pears. However, a progressive loss of culturability on solid media by 2 to 3 logarithmic units during storage time was observed, the rate at which cells became nonculturable dependent on the type of water used. A significant difference in the time to nonculturability between cells maintained in irrigation water and those kept in deionized water was observed, associated with the content of dissolved compounds, which differ for these two types of water. Since bacterial cells maintained their viability, it seems that the oligotrophic conditions found in natural aquatic environments could allow the survival of the fireblight pathogen. Further, the maintenance of the pathogenicity of E. amylovora supports the possible role of water as a reservoir and vehicle for transmission of this pathogen.

Summary The eel and human pathogen Vibrio vulnificus serovar E (biotype 2) is seldom isolated from natural waters, although it can survive in sterilized artificial seawater microcosms for years. The main objective of the present study was to investigate whether aquatic microbiota can limit its survival and recovery from water samples. A set of preliminary experiments of survival in microcosms containing natural seawater and water from eel farms showed that the persistence of this pathogen was mainly controlled by grazing, and secondarily by bacterial competition. The bacte- rial competition was further analysed in artificial seawater microcosms co-inoculated with selected virulent serovar E (VSE) strains and potential compet- itors. Competitors included V. vulnificus biotype 1 iso- lates and strains of selected species that can grow on the selective media designed for V. vulnificus isola- tion from water samples. Evidences of bacterial com- petition that was detrimental for VSE recovery were recorded. Thus, some species produced a deleterious effect on VSE strains under starvation, and others were able to use the resources more efficiently under nutrient input. These results suggest that an over- growth of more efficient competitor bacteria in con- ventional media used for isolation of V. vulnificus could mask the recovery of VSE strains and explain the scarcity of reports on the isolation of this human and eel pathogen from natural waters.

Vibrio vulnificus serovar E (formerly biotype 2) is the etiologic agent that is responsible for the main infectious disease affecting farmed eels. Although the pathogen can theoretically use water as a vehicle for disease transmission, it has not been isolated from tank water during epizootics to date. In this work, the mode of transmission of the disease to healthy eels, the portals of entry of the pathogen into fish, and their putative reservoirs have been investigated by means of laboratory and field experiments. Results of the experiments of direct and indirect host-to-host transmission, patch contact challenges, and oral-anal intubations suggest that water is the prime vehicle for disease transmission and that gills are the main portals of entry into the eel body. The pathogen mixed with food can also come into the fish through the gastrointestinal tract and develop the disease. These conclusions were supported by field data obtained during a natural outbreak in which we were able to isolate this microorganism from tank water for the first time. The examination of some survivors from experimental infections by indirect immunofluorescence and scanning electron microscopy showed that V. vulnificus serovar E formed a biofilm-like structure on the eel skin surface. In vitro assays demonstrated that the ability of the pathogen to colonize both hydrophilic and hydrophobic surfaces was inhibited by glucose. The capacity to form biofilms on eel surface could constitute a strategy for surviving between epizootics or outbreaks, and coated survivors could act as reservoirs for the disease.

The applications of an indirect fluorescent antibody technique (IFAT), developed to detect and enumerate the pathogenic bacterium Vibrio vulnificus serovar E from water and clinical samples, are described. This technique proved accurate for detecting V. vulnificus, even under starvation conditions and in the non-culturable state, and could differentiate this species from other bacteria which share the same habitats. The IFAT was successfully used to diagnose vibriosis from naturally- and artificially-infected eels. The overall data suggest that applying this technique properly in environmental and epidemiological/epizootiological studies could significantly increase our knowledge of this bacterium.

The existence of strains of Vibrio vulnificus serovar E that are avirulent for eels is reported in this work. These isolates were recovered from water and oysters and differed from eel virulent strains in (i) fermentation and utilization of mannitol, (ii) ribotyping after Hin dIII digestion, and (iii) susceptibility to eel serum. Lipopolysaccharide of these strains lacked the highest molecular weight immunoreactive bands, which are probably involved in serum resistance.

The survival of fish-virulent strains of Photobacterium damselae subsp. damselae in seawater microcosms, with and without sediment, was investigated. The strains survived as culturable bacteria at 14 and 22 degrees C for at least 1 year, and infectivity for fish was maintained. At 5 degrees C, cells lost culturability on solid media, but this was recovered when the temperature was increased to 22 degrees C. Finally, morphological changes in the bacterium (rod to coccus), and production of vesicles and extracellular material were observed during the time of starvation. The overall results suggest that seawater and sediment can act as reservoirs for these virulent strains.

Little is known about the survival mechanisms of Erwinia amylovora outside its hosts. It has been demonstrated that it enters the viable but nonculturable state (VBNC) when exposed to different types of stress. In the VBNC state, bacterial cells remain viable but unable to grow on the solid general media where they usually do, and are thus undetectable by conventional culture-dependent methods. In this work, we have evaluated the recovery of E. amylovora VBNC cells by passage through pear plantlets, in comparison with other recovery methods commonly used for this pathogen: incubation in KB broth and inoculation of immature fruits. VBNC cells were obtained by exposure of bacterial cells to different types of stress (oligotrophy, nutrient deprivation and chlorine), and recovery assays were performed at 26 degrees C. In all cases, the recovery of VBNC cells was more effective in plantlets than in liquid KB or immature fruits. In fact, when cells were exposed to chlorine for more than 30 min, only passage through host plant gave positive result, enabling recovery of E. amylovora cells few days after inoculation of plants. These results suggest a higher effectiveness of in planta recovery than those performed with liquid KB or detached fruits. Our results support the hypothesis of the VBNC state being part of the E. amylovora life cycle. The potential existence of this physiological state in nature should be taken in consideration in epidemiological studies of fire blight, with the aim to optimize the management and control of this disease.

Rhizobium rhizogenes strain K84 is a commercial biocontrol agent used worldwide to control crown gall disease. The organism binds tightly to polypropylene substrate and efficiently colonizes root surfaces as complex, multilayered biofilms. A genetic screen identified two mutants in which these surface interactions were affected. One of these mutants failed to attach and form biofilms on the abiotic surface although, interestingly, it exhibited normal biofilm formation on the biological root tip surface. This mutant is disrupted in a wcbD ortholog gene, which is part of a large locus predicted to encode functions for the biosynthesis and export of a group II capsular polysaccharide (CPS). Expression of a functional copy of wcbD in the mutant background restored the ability of the bacteria to attach and form normal biofilms on the abiotic surface. The second identified mutant attached and formed visibly denser biofilms on both abiotic and root tip surfaces. This mutant is disrupted in the rkpK gene, which is predicted to encode a UDP-glucose 6-dehydrogenase required for O-antigen lipopolysaccharide (LPS) and K-antigen capsular polysaccharide (KPS) biosynthesis in rhizobia. The rkpK mutant from strain K84 was deficient in O-antigen synthesis and exclusively produced rough LPS. We also show that strain K84 does not synthesize the KPS typical of some other rhizobia strains. In addition, we identified a putative type II CPS, distinct from KPS, that mediates cell-surface interactions, and we show that O antigen of strain K84 is necessary for normal cell-cell interactions in the biofilms.

Lucas-Elió, P. et al., Five novel Gram-reaction-negative aerobic marine bacterial strains with DNA G+C contents