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Erwinia amylovora causes fire blight, a destructive disease of rosaceous plants very difficult to control. We demonstrated that copper, employed to control plant diseases, induces the "viable-but-nonculturable" (VBNC) state in E. amylovora. Moreover, it was previously reported that copper increases production of its main exopolysaccharide (EPS), amylovoran. In this work, the copper-complexing ability of amylovoran and levan, other major EPS of E. amylovora, was demonstrated. Following this, EPS-deficient mutants were used to determine the role of these EPSs in survival of this bacterium in AB mineral medium with copper, compared to their wild type strain and AB without copper. Total, viable and culturable counts of all strains were monitored for six months. With copper, a larger fraction of the viable population of EPS mutants entered into the VBNC state, and earlier than their wild type strain, showing the contribution of both EPSs to long-term survival in a culturable state. Further, we demonstrated that both EPSs can be used as carbon source by E. amylovora under deprivation conditions. Overall, these previously unreported functions of amylovoran and levan provide survival advantages for E. amylovora, which could contribute to its enhanced persistence in nature. (C) 2010 Elsevier Masson SAS. All rights reserved.

Novel aerobic, Gram-negative bacteria with DNA G+C contents below 50 mol% were isolated from the culturable microbiota associated with the Mediterranean seagrass Posidonia oceanica. 16S rRNA gene sequence analyses revealed that they belong to the genus Marinomonas. Strain IVIA-Po-186 is a strain of the species Marinomonas mediterranea, showing 99.77% 16S rRNA gene sequence similarity with the type strain, MMB-1T, and sharing all phenotypic characteristics studied. This is the first description of this species forming part of the microbiota of a marine plant. A second strain, designated IVIA-Po-101T, was closely related to M. mediterranea based on phylogenetic studies. However, it differed in characteristics such as melanin synthesis and tyrosinase, laccase and antimicrobial activities. In addition, strain IVIA-Po-101T was auxotrophic and unable to use acetate. IVIA-Po-101T shared 97.86% 16S rRNA gene sequence similarity with M. mediterranea MMB-1T, but the level of DNA-DNA relatedness between the two strains was only 10.3%. On the basis of these data, strain IVIA-Po-101T is considered to represent a novel species of the genus Marinomonas, for which the name Marinomonas balearica sp. nov. is proposed. The type strain is IVIA-Po-101T (=CECT 7378T =NCIMB 14432T). A third novel strain, IVIA-Po-185T, was phylogenetically distant from all recognized Marinomonas species. It shared the highest 16S rRNA gene sequence similarity (97.4%) with the type strain of Marinomonas pontica, but the level of DNA-DNA relatedness between the two strains was only 14.5%. A differential chemotaxonomic marker of this strain in the genus Marinomonas is the presence of the fatty acid C17:0 cyclo. Strain IVIA-Po-185T is thus considered to represent a second novel species of the genus, for which the name Marinomonas pollencensis sp. nov. is proposed. The type strain is IVIA-Po-185T (=CECT 7375T =NCIMB 14435T). An emended description of the genus Marinomonas is given based on the description of these two novel species, as well as other Marinomonas species described after the original description of the genus. © 2010 IUMS., This study was partially supported by grant 03026/PI/05 from the Seneca Foundation, Autonomous Government of the Region of Murcia, and by a grant of Fundación BBVA.

E.G. Biosca , R.D. Santander, M. Ordax, E. Marco-Noales, B. Aguila, A. Flores and M.M. Lopez 1 Universidad de Valencia, Departamento de Microbiologia y Ecologia, Avenida Dr. Moliner 50, 46100, Burjassot, Valencia, Spain. 2 Instituto Valenciano de Investigaciones Agrarias (IVIA), Centro de Proteccion Vegetal y Biotecnologia, Carretera Moncada – Naquera, km 4.5, 46113, Moncada,Valencia, Spain. 3 Universidad de Valencia, Servicio Central de Soporte a la Investigacion Experimental, Avenida Dr. Moliner 50, 46100, Burjassot, Valencia, Spain.

Aims: Survival of Erwinia amylovora, causal agent of fire blight in pome fruits and other rosaceous plants, was monitored inside mature apples calyces under some storage conditions utilized in fruit. Methods and Results: Apple fruit calyces inoculated with two E. amylovora strains and their respective GFP-marked strains were maintained at 26° and 5°C, and the effect of copper treatment was assayed at 0·01 and 0·1 mmol l−1 CuSO4. In nontreated apples at 26°C, part of the population of E. amylovora survived in the ‘viable but nonculturable’ (VBNC) state, whereas at 5°C the majority of the population retained culturability. In copper-treated apples, the whole population adopted the VBNC state irrespective of temperature. Regardless of temperature, copper and inoculum dose, VBNC cells recovered culturability and pathogenicity in King’s B broth or by host plant passage. Conclusions: Erwinia amylovora survived for at least 35 days in mature apple calyces. Besides, the ability of the pathogen in the VBNC state to regain culturability and pathogenicity suggests that the apple fruit could be a potential carrier of E. amylovora contributing to the spreading of fire blight disease. Significance and Impact of the Study: The risk of E. amylovora dissemination through mature fruit transport, although low, has been demonstrated, and should be considered in pest risk assessments.

Marco-Noales, E., Bertolini, E., Morente, C., and Lopez, M. M. 2008. Integrated approach for detection of nonculturable cells of Rastonia solanacearum in asymptomatic Pelargonium spp. cuttings. Phytopathology 98:949-955. Ralstonia solanacearum (biovar 2, race 3) is a soil and water-borne pathogen that causes serious diseases in several solanaceous hosts. It can also infect geranium plants, posing an important threat to their culture when latently infected cuttings are imported from countries where the pathogen is endemic. R. solanacearum can be present in very low numbers in asymptomatic geranium cuttings, and/or in a particular stressed physiological state that escapes direct isolation on the solid media usually employed. Consequently, an integrated protocol has been developed to analyze asymptomatic geranium cuttings routinely. The first screening tests include isolation and co-operational-polymerase chain reaction (CoPCR), based on the simultaneous and co-operational action of three primers from 16S rRNA of R. solanacearum. This method was selected as the most sensitive one, able to detect only 1 cell/ml including nonculturable cells. When isolation is negative but Co-PCR is positive, the bioassay in tomato plants is proposed, since stressed bacterial cells or those present in low numbers that do not grow on solid media can be recovered from inoculated tomato plants and retain pathogenicity. This methodology has been demonstrated to be useful and has allowed us to assess the relevance of the physiological status of bacterial cells and its implications in detection. It also reveals the risk of introducing R. solanacearum through asymptomatic geranium material when relying only on bacterial isolation.

Genetically modified potatoes expressing antibacterial protein T4 lysozyme may offer effective control strategies for bacterial pathogens causing severe potato diseases. Apart from this beneficial effect, it is very important to investigate such engineered potatoes carefully for potential adverse effects on potato-associated bacteria which frequently exhibit plant beneficial functions such as plant growth promotion and antagonism towards pathogens invading the plant. Two field experiments were carried out in Spain to analyze the potential effects of conventional and genetically modified T4-lysozyme producing potatoes on shoot-associated bacteria. The first baseline field trial 2002 was performed in Meliana in which three conventional potato lines, Achirana Inta, Desiree, and Merkur, were cultivated and sampled at flowering. The second field trial was conducted in Cella in 2003 in order to compare the effects of a senescent transgenic, T4 lysozyme expressing potato trait, Desiree DL 12, with its isogenic, non-transformed parental line Desiree. Structural characteristics of potato shoot-associated bacteria was assayed by 16S rRNA-based terminal restriction fragment length polymorphism (T-RFLP) analysis and dominant community members within T-RFLP profiles were identified by sequence analysis of generated 16S rRNA gene libraries. Cultivable bacteria isolated from shoots of potatoes grown in the Meliana field trial were monitored for antibiosis against Ralstonia solanacearum, whereas isolates derived from shoots of potatoes cultivated in the Cella trial were screened for antagonism against Ralstonia solanacearum and Rhizoctonia solani, and for 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase production. Determined antagonists were identified by 16S rRNA gene analysis. All potato traits hosted a cultivar-specific community of bacteria with antagonism against the pathogens and/or potential to produce ACC deaminase. Several antagonists obtained from the Cella field potatoes were also observed as ACC deaminase producers. Community profiling revealed a greater diversity differentiation between the senescent T4 lysozyme expressing and parental Desiree lines grown in the Cella field as compared to the variations between the three flowering conventional lines cultivated in the Meliana field trial. Effects of the two varying field sites and different vegetation stages were greater than those of T4 lysozyme when investigating the community composition of bacteria colonizing the shoots of the Desiree line cultivated in both field trials.

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 bacterial competition was further analysed in artificial seawater microcosms co-inoculated with selected virulent serovar E (VSE) strains and potential competitors. Competitors included V. vulnificus biotype 1 isolates and strains of selected species that can grow on the selective media designed for V. vulnificus isolation from water samples. Evidences of bacterial competition 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 overgrowth of more efficient competitor bacteria in conventional 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 biotype 2 is a primary eel pathogen which constitutes a lipopolysaccharide (LPS)-based homogeneous O serogroup within the species. In the present work, we have developed an enzyme-linked immunosorbent assay (ELISA) based on the specificity of LPS for the detection of this pathogen. The ELISA specificity was confirmed after testing 36 biotype 2 strains from laboratory cultures and environmental samples, 31 clinical and environmental biotype 1 isolates, and several strains of Vibrio, Aeromonas, and Yersinia species, including the fish pathogens V. anguillarum, V. furnissii, A. hydrophila, and Y. ruckerii. The detection limits for biotype 2 cells were around 10(4) to 10(5) cells/well, and the immunoassay was also able to detect cells in the nonculturable state. Artificially infected eels and environmental samples were analyzed, and the immunodetection was confirmed by cultural methods (isolation on selective and nonselective media before and after broth enrichment). With this methodology, V. vulnificus biotype 2 was successfully detected in infected eels and asymptomatic carriers, which suggests that eels can act as a reservoir for this pathogen.

Xylella fastidiosa is an economically important plant pathogenic bacterium of global importance associated, since 2013, with a devastating epidemic in olive trees in Italy. Since then, several outbreaks of this pathogen have been reported in other European member countries including Spain, France, and Portugal. In Spain, the three major subspecies (subsp. fastidiosa, multiplex, and pauca) of the bacterium have been detected in the Balearic Islands, but only subspecies multiplex in the mainland (Alicante). We present the first complete genome sequences of two Spanish strains: X. fastidiosa subsp. fastidiosa IVIA5235 from Mallorca and X. fastidiosa subsp. multiplex IVIA5901 from Alicante, using Oxford Nanopore and Illumina sequence reads, and two hybrid approaches for genome assembly. These completed genomes will provide a resource to better understand the biology of these X. fastidiosa strains., 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 POnTE (Pest Organisms Threatening Europe grant 635646), and Desarrollo de estrategias de erradicación, contención y control de Xylella fastidiosa 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 and COST Action CA16107 EuroXanth supported by European Cooperation in Science and Technology.e

At present, no reports exist on the isolation of the eel pathogen Vibrio vulnificus biotype 2 from water samples. Nevertheless, it has recently been demonstrated that this biotype can use water as a route of infection. In the present study, the survival of this pathogen in artificial seawater (ASW) microcosms at different temperatures (25 and 5 degrees C) was investigated during a 50-day period, with biotype 1 as a control, V. vulnificus biotype 2 was able to survive in the culturable state in ASW at 25 degrees C in the free-living form, at least for 50 days, entering into the nonculturable state when exposed to low temperature. In this state, this microorganism survived with reduced rates of activity, showing marked changes in size and morphology. The rate at which cells became nonculturable was dependent on their physiological age. The capsule seems not to be necessary for the survival of biotype 2 in aquatic environments as a free-living organism. Culturability remained the highest on modified salt water yeast extract agar, which is closer in salt and nutrient composition to ASW than heart infusion agar. Biotype 2 cells recovered culturability on solid media after an increase of incubation temperature from 5 to 25 degrees C. Culturable cells of this bacterium maintained infectivity for either eel or mice, while dormant cells seemed to lose their virulence. The former finding suggests that the aquatic environment is a reservoir and vehicle of transmission of this pathogen.

The emergence of Xylella fastidiosa in Europe revealed the occurrence of bacterial strains belonging to different subspecies and unraveled the vulnerability of some typical European plant species. Among these, olive was found infected in different European outbreaks, with infections caused by strains harboring different sequence types (STs) and belonging to subspecies pauca and multiplex. In this work, we compared under greenhouse conditions, the response of the olive cultivar “Cellina di Nardò” to infections caused by the strains De Donno (subsp. pauca, ST53) and ESVL (subsp. multiplex, ST6), as this cultivar is one of the most susceptible in the Italian outbreak. The data showed that: (i) strain De Donno systemically colonized the plants faster than the strain ESLV; (ii) 1.5 years post-inoculation, the bacterial population detected in the plants systemically infected was ~25 times higher for strain De Donno than ESLV; (iii) a severe shoot dieback were detected on the De Donno-infected plants, whereas up to 3 years post-inoculation no dieback and desiccations have been recorded on the ESLV-infected plants. This pathogenicity test was complemented with an RNA-seq analysis. Nine libraries, three for each condition (ESVL, De Donno, mock inoculated), were sequenced by Ilumina technology and sequences mapped against the Olea europeae cultivar Picual genome for the analysis of differential gene expression (DEG). The output of this study showed that, respect to the 6,200 genes differentially expressed (FDR The present data showed that strains of the two subspecies elicit differential host responses, which were biologically and molecularly attenuated in the ESVL infections with respect to the aggressive De Donno infections and provide useful insights for exploring the molecular basis of Xylella-olive interaction.

Proceedings of AgEng 2018, Wageningen (The Netherlands) 8-11 July 2018

Vibrio vulnificus is a gram-negative bacterium capable of producing septicemic infections in eels and immunocompromised humans. Two biotypes are classically recognized, with the virulence for eels being specific to strains belonging to biotype 2, which constitutes a homogeneous lipopolysaccharide (LPS)-based O serogroup (which we have designated serogroup E). In the present study we demonstrated that the O side chain of this LPS determines the selective virulence of biotype 2 for eels: (i) biotype 1 strains (which do not belong to serogroup E) are destroyed by the bactericidal action of nonimmune eel serum (NIS) through activation of the alternative pathway of complement, (ii) biotype 2 strains (of serogroup E) are resistant to NIS, and (iii) rough mutants of biotype 2 lacking the O polysaccharide side chain are sensitive to NIS and avirulent for eels.

Xylella fastidiosa está considerada como la bacteria que constituye la principal amenaza hoy día para distintos cultivos de gran importancia estratégica, en todos los países donde aún no está presente. Por ello, es una bacteria de cuarentena en la Unión Europea (UE) desde el año 2000, según la Directiva 2000/29/EC, y también está incluida en la lista A1 de la Organización Europea y Mediterránea de Protección de Plantas (EPPO) (http://www.eppo.org/ QUARANTINE/listA1.htm). Su peligro radica en que es el agente causal de numerosas enfermedades muy graves, que podrían tener enormes repercusiones económicas para distintos cultivos europeos y muy especialmente para la economía española, dado que puede afectar a olivo, vid, cítricos, frutales de hueso, almendro y numerosas especies ornamentales y forestales.

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., ES; PPT; marco_est@gva.es

Vibrio vulnificus biotype 2 (serovar E) is a primary eel pathogen. In this study, we performed long-term survival experiments to investigate whether the aquatic ecosystem can be a reservoir for this bacterium. We have used microcosms containing water of different salinities (ranging from 0.3 to 3.8%) maintained at three temperatures (12, 25, and 30°C). Temperature and salinity significantly affected long-term survival: (i) the optimal salinity for survival was 1.5%; (ii) lower salinities reduced survival, although they were nonlethal; and (ii) the optimal temperature for survival was dependent on the salinity (25°C for microcosms at 0.3 and 0.5% and 12°C for microcosms at 1.5 to 3.8%). In the absence of salts, culturability dropped to zero in a few days, without evidence of cellular lysis. Under optimal conditions of salinity and temperature, the bacterium was able to survive in the free-living form for at least 3 years. The presence of a capsule on the bacterial cell seemed to confer an advantage, since the long-term survival rate of opaque variants was significantly higher than that of translucent ones. Long-term-starved cells maintained their infectivity for eels (as determined by both intraperitoneal and immersion challenges) and mice. Examination under the microscope showed that (i) the capsule was maintained, (ii) the cell size decreased, (iii) the rod shape changed to coccuslike along the time of starvation, and (iv) membrane vesicles and extracellular material were occasionally produced. In conclusion, V. vulnificus biotype 2 follows a survival strategy similar to that of biotype 1 of this species in response to starvation conditions in water. Moreover, the aquatic ecosystem is one of its reservoirs.