Complete Circularized Genome Resources of Seven Strains of Xylella fastidiosa subsp. fastidiosa Using Hybrid Assembly Reveals Unknown Plasmids.

Xylella fastidiosa 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 Oxford Nanopore Technologies and Illumina sequencing data, we assembled high-quality complete genomes of seven X. fastidiosa subsp. fastidiosa strains isolated from different plants in Spain, the United States, and Mexico. Comparative genomic analyses discovered differences in plasmid content among strains, including plasmids that had been overlooked previously when using the Illumina sequencing platform alone. Interestingly, in strain CFBP8073, intercepted in France from plants imported from Mexico, three plasmids were identified, including two (plasmids 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 X. fastidiosa 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 United States. 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.

First report of bacterial leaf blight caused by Xanthomonas hortorum pv. carotae on carrots in Spain.

In September 2019, symptoms resembling those of bacterial leaf blight were observed on carrot plants (Daucus carota L. subsp. sativus Hoffm.) cv. Romance cultivated in commercial plots in Chañe (Segovia), Spain. Symptoms were observed in two plots surveyed representing three hectares, with an incidence greater than 90%, and also in some plots in other nearby municipalities sown with the same batch of seeds. The lesions observed at the ends of the leaves were initially yellow that develop dark brown to black with chlorotic halos on leaflets that turned necrotic. Yellow, Xanthomonas-like colonies were isolated onto YPGA medium (Ridé 1969) from leaf lesions. Two bacterial isolates were selected and confirmed by real-time PCR using a specific primer set for Xanthomonas hortorum pv. carotae (Temple et al. 2013). All isolates were gram-negative, aerobic rods positive for catalase, able of hydrolyzing casein and aesculin and growing at 2% NaCl, while were negative for oxidase and urease tests. Sequences of 16S rRNA gene showed 100% similarity with Xanthomonas campestris, X. arboricola, X. gardneri, X. cynarae strains (GenBank accession numbers: MW077507.1 and MW077508.1 for the isolates CRD19-206.3 and CRD19-206.4, respectively). However, the resulting phylogeny of multilocus sequence analysis (MLSA) of a concatenation of the housekeeping genes atpD, dnaK, and efp (Bui Thi Ngoc et al. 2010), by using neighbour-joining trees generated with 500 bootstrap replicates, grouped the two isolates with the X. hortorum pv. carotae M081 strain (Kimbrel et al. 2011) (GenBank accession numbers: MW161270 and MW161271 for atpD for the two isolates, respectively; MW161268 and MW161269 for dnaK; MW161272 and MW161273 for efp). A pairwise identity analysis revealed a 100% identity between all three isolates. Pathogenicity of the isolates was tested by spray inoculation (Christianson et al. 2015) with a bacterial suspension (108 CFU/ml) prepared in sterile distilled water at 3 to 4 true-leaf stage (six plants per isolate). Sterile distilled water was used as negative control. The inoculated plants were incubated in a growth chamber (25°C and 95% relative humidity [RH]) for 72 h, and then transferred to a greenhouse at 24 to 28°C and 65% RH. Characteristic leaf blight symptoms developed on inoculated carrot plants, while no symptoms were observed on the negative control plants 20 days after inoculation. The bacterium was re-isolated from symptomatic tissue and the identity confirmed through PCR analysis. Based on PCR, morphological and phenotypic tests, sequence analysis, and pathogenicity assays, the isolates were identified as X. hortorum pv. carotae. To our knowledge, this is the first report of bacterial leaf blight of carrot caused by X. hortorum pv. carotae in Spain, and the first molecular and pathological characterization. It is important to early detect this pathogen and take suitable measures to prevent its spread, since it could cause yield losses for a locally important crop such as carrot.

Emergence of a Plant Pathogen in Europe Associated with Multiple Intercontinental Introductions.

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.IMPORTANCEXylella fastidiosa is an economically important plant-pathogenic bacterium that has emerged as a pathogen of global importance associated with a devastating epidemic in olive trees in Italy associated with X. fastidiosa subspecies pauca and other outbreaks in Europe, such as X. fastidiosa subspecies fastidiosa and X. fastidiosa subspecies multiplex in Spain and X. fastidiosa subspecies multiplex in France. We present evidence of multiple introductions of X. fastidiosa subspecies multiplex, likely from the United States, into Spain, Italy, and France. These introductions illustrate the risks associated with the commercial trade of plant material at global scales and the need to develop effective policy to limit the likelihood of pathogen pollution into naive regions. Our study demonstrates the need to utilize whole-genome sequence data to study X. fastidiosa introductions at outbreak stages, since a limited number of genetic markers does not provide sufficient phylogenetic resolution to determine dispersal paths or relationships among strains that are of biological and quarantine relevance.

Complete Circularized Genome Data of Two Spanish strains of Xylella fastidiosa (IVIA5235 and IVIA5901) Using Hybrid Assembly Approaches.

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.

Draft Genome Resources of Two Strains ("ESVL" and "IVIA5901") of Xylella fastidiosa Associated with Almond Leaf Scorch Disease in Alicante, Spain.

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.

Xanthomonas prunicola sp. nov., a novel pathogen that affects nectarine (Prunus persica var. nectarina) trees.

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).

Real-time on-site detection of the three 'Candidatus Liberibacter' species associated with HLB disease: a rapid and validated method.

Huanglongbing (HLB) is a devastating disease that affects all commercial citrus species worldwide. The disease is associated with bacteria of three species of the genus 'Candidatus Liberibacter' transmitted by psyllid vectors. To date, HLB has no cure, so preventing its introduction into HLB-free areas is the best strategy to control its spread. For that, the use of accurate, sensitive, specific, and reliable detection methods is critical for good integrated management of this serious disease. This study presents a new real-time recombinase polymerase amplification (RPA) protocol able to detect the three 'Ca. Liberibacter' species associated with HLB in both plant and insect samples, validated according to European and Mediterranean Plant Protection Organization (EPPO) guidelines and tested on 365 samples from nine different geographic origins. This new protocol does not require nucleic acid purification or specialized equipment, making it ideal to be used under field conditions. It is based on specific primers and probe targeting a region of fusA gene, which shows a specificity of 94%-100%, both in silico and in vitro, for the 'Ca. Liberibacter' species associated with HLB. The analytical sensitivity of the new protocol is excellent, with a reliable detection limit in the order of 101 copies per microliter in HLB-infected plant and insect material. The repeatability and reproducibility of the new methods showed consistent results. Diagnostic parameters of the new RPA protocol were calculated and compared with the gold standard technique, a quantitative real-time PCR, in both crude extracts of citrus plants and insect vectors. The agreement between the two techniques was almost perfect according to the estimated Cohen's kappa index, with a diagnostic sensitivity and specificity of 83.89% and 100%, respectively, and a relative accuracy of 91.59%. Moreover, the results are obtained in less than 35 min. All these results indicate the potential of this new RPA protocol to be implemented as a reliable on-site detection kit for HLB due to its simplicity, speed, and portability.

A new and accurate qPCR protocol to detect plant pathogenic bacteria of the genus 'Candidatus Liberibacter' in plants and insects.

Four pathogenic bacterial species of the genus 'Candidatus Liberibacter', transmitted by psyllid vectors, have been associated with serious diseases affecting economically important crops of Rutaceae, Apiaceae and Solanaceae families. The most severe disease of citrus plants, huanglongbing (HLB), is associated with 'Ca. Liberibacter asiaticus' (CaLas), 'Ca. Liberibacter americanus' (CaLam) and 'Ca. Liberibacter africanus' (CaLaf), while 'Ca. Liberibacter solanacearum' (CaLsol) is associated with zebra chip disease in potatoes and vegetative disorders in apiaceous plants. Since these bacteria remain non-culturable and their symptoms are non-specific, their detection and identification are done by molecular methods, mainly based on PCR protocols. In this study, a new quantitative real-time PCR protocol based on TaqMan probe, which can also be performed in a conventional PCR version, has been developed to detect the four known phytopathogenic species of the genus Liberibacter. The new protocol has been validated according to European Plant Protection Organization (EPPO) guidelines and is able to detect CaLas, CaLam, CaLaf and CaLsol in both plants and vectors, not only using purified DNA but also using crude extracts of potato and citrus or psyllids. A comparative analysis with other previously described qPCR protocols revealed that this new one developed in this study is more specific and equally or more sensitive. Thus, other genus-specific qPCR protocols have important drawbacks regarding the lack of specificity, while with the new protocol there was no cross-reactions in 250 samples from 24 different plant and insect species from eight different geographical origins. Therefore, it can be used as a rapid and time-saving screening test, as it allows simultaneous detection of all plant pathogenic species of 'Ca. Liberibacter' in a one-step assay.

Role for Rhizobium rhizogenes K84 cell envelope polysaccharides in surface interactions.

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.

Plant-associated microbiota as a source of antagonistic bacteria against the phytopathogen Erwinia amylovora.

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.

Xylella fastidiosa Infection Reshapes Microbial Composition and Network Associations in the Xylem of Almond Trees.

Xylella fastidiosa represents a major threat to important crops worldwide including almond, citrus, grapevine, and olives. Nowadays, there are no efficient control measures for X. fastidiosa, and the use of preventive measures and host resistance represent the most practical disease management strategies. Research on vessel-associated microorganisms is gaining special interest as an innate natural defense of plants to cope against infection by xylem-inhabiting pathogens. The objective of this research has been to characterize, by next-generation sequencing (NGS) analysis, the microbial communities residing in the xylem sap of almond trees affected by almond leaf scorch disease (ALSD) in a recent X. fastidiosa outbreak occurring in Alicante province, Spain. We also determined community composition changes and network associations occurring between xylem-inhabiting microbial communities and X. fastidiosa. For that, a total of 91 trees with or without ALSD symptoms were selected from a total of eight representative orchards located in five municipalities within the X. fastidiosa-demarcated area. X. fastidiosa infection in each tree was verified by quantitative polymerase chain reaction (qPCR) analysis, with 54% of the trees being tested X. fastidiosa-positive. Globally, Xylella (27.4%), Sphingomonas (13.9%), and Hymenobacter (12.7%) were the most abundant bacterial genera, whereas Diplodia (30.18%), a member of the family Didymellaceae (10.7%), and Aureobasidium (9.9%) were the most predominant fungal taxa. Furthermore, principal coordinate analysis (PCoA) of Bray-Curtis and weighted UniFrac distances differentiated almond xylem bacterial communities mainly according to X. fastidiosa infection, in contrast to fungal community structure that was not closely related to the presence of the pathogen. Similar results were obtained when X. fastidiosa reads were removed from the bacterial data set although the effect was less pronounced. Co-occurrence network analysis revealed negative associations among four amplicon sequence variants (ASVs) assigned to X. fastidiosa with different bacterial ASVs belonging to 1174-901-12, Abditibacterium, Sphingomonas, Methylobacterium-Methylorubrum, Modestobacter, Xylophilus, and a non-identified member of the family Solirubrobacteraceae. Determination of the close-fitting associations between xylem-inhabiting microorganisms and X. fastidiosa may help to reveal specific microbial players associated with the suppression of ALSD under high X. fastidiosa inoculum pressure. These identified microorganisms would be good candidates to be tested in planta, to produce almond plants more resilient to X. fastidiosa infection when inoculated by endotherapy, contributing to suppress ALSD.

Marinomonas alcarazii sp. nov., M. rhizomae sp. nov., M. foliarum sp. nov., M. posidonica sp. nov. and M. aquiplantarum sp. nov., isolated from the microbiota of the seagrass Posidonia oceanica.

Five novel Gram-reaction-negative aerobic marine bacterial strains with DNA G+C contents <50 mol% were isolated from the seagrass Posidonia oceanica. 16S rRNA sequence analysis indicated that they belonged to the genus Marinomonas. Major fatty acid compositions, comprising C10:0 3-OH, C16:0, C16:1ω7c and C18:1ω7c, supported the affiliation of these strains to the genus Marinomonas. Strains IVIA-Po-14b(T), IVIA-Po-145(T) and IVIA-Po-155(T) were closely related to Marinomonas pontica 46-16(T), according to phylogenetic analysis. However, DNA-DNA hybridization values <35 % among these strains revealed that they represented different species. Further differences in the phenotypes and minor fatty acid compositions were also found among the strains. Another two strains, designated IVIA-Po-181(T) and IVIA-Po-159(T), were found to be closely related to M. dokdonensis DSW10-10(T) but DNA-DNA relatedness levels <40 % in pairwise comparisons, as well as some additional differences in phenotypes and fatty acid compositions supported the creation of two novel species. Accordingly, strains IVIA-Po-14b(T )( = CECT 7730(T)  = NCIMB 14671(T)), IVIA-Po-145(T) ( = CECT 7377(T)  = NCIMB 14431(T)), IVIA-Po-155(T) ( = CECT 7731(T)  = NCIMB 14672(T)), IVIA-Po-181(T) ( = CECT 7376(T)  = NCIMB 14433(T)) and IVIA-Po-159(T) ( = CECT 7732(T)  = NCIMB 14673(T)) represent novel species, for which the names Marinomonas alcarazii sp. nov., Marinomonas rhizomae sp. nov., Marinomonas foliarum sp. nov., Marinomonas posidonica sp. nov. and Marinomonas aquiplantarum sp. nov. are proposed, respectively.

Transcriptional response of Erwinia amylovora to copper shock: in vivo role of the copA gene.

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.

Draft Genome Sequence of Xylella fastidiosa subsp. fastidiosa Strain IVIA5235, Isolated from Prunus avium in Mallorca Island, Spain.

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.

Lateral flow immunoassay for on-site detection of Xanthomonas arboricola pv. pruni in symptomatic field samples.

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 104 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.

Genetic diversity reflects geographical origin of Ralstonia solanacearum strains isolated from plant and water sources in Spain.

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.

Medfly Ceratitis capitata as Potential Vector for Fire Blight Pathogen Erwinia amylovora: Survival and Transmission.

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.

Exopolysaccharides favor the survival of Erwinia amylovora under copper stress through different strategies.

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.

Taxonomic study of Marinomonas strains isolated from the seagrass Posidonia oceanica, with descriptions of Marinomonas balearica sp. nov. and Marinomonas pollencensis sp. nov.

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-1(T), 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-101(T), 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-101(T) was auxotrophic and unable to use acetate. IVIA-Po-101(T) shared 97.86 % 16S rRNA gene sequence similarity with M. mediterranea MMB-1(T), but the level of DNA-DNA relatedness between the two strains was only 10.3 %. On the basis of these data, strain IVIA-Po-101(T) 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-101(T) (=CECT 7378(T) =NCIMB 14432(T)). A third novel strain, IVIA-Po-185(T), 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 C(17 : 0) cyclo. Strain IVIA-Po-185(T) 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-185(T) (=CECT 7375(T) =NCIMB 14435(T)). 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.

Are molecular tools solving the challenges posed by detection of plant pathogenic bacteria and viruses?

Plant pathogenic bacteria, phytoplasmas, viruses and viroids are difficult to control, and preventive measures are essential to minimize the losses they cause each year in different crops. In this context, rapid and accurate methods for detection and diagnosis of these plant pathogens are required to apply treatments, undertake agronomic measures or proceed with eradication practices, particularly for quarantine pathogens. In recent years, there has been an exponential increase in the number of protocols based on nucleic-acid tools being those based on PCR or RT-PCR now routinely applied worldwide. Nucleic acid extraction is still necessary in many cases and in practice inhibition problems are decreasing the theoretical sensitivity of molecular detection. For these reasons, integrated protocols that include the use of molecular techniques as screening methods, followed by confirmation by other techniques supported by different biological principles are advisable. Overall, molecular techniques based on different types of PCR amplification and very especially on real-time PCR are leading to high throughput, faster and more accurate detection methods for the most severe plant pathogens, with important benefits for agriculture. Other technologies, such as isothermal amplification, microarrays, etc. have great potential, but their practical development in plant pathology is still underway. Despite these advances, there are some unsolved problems concerning the detection of many plant pathogens due to their low titre in the plants, their uneven distribution, the existence of latent infections and the lack of validated sampling protocols. Research based on genomic advances and innovative detection methods as well as better knowledge of the pathogens' lifecycle, will facilitate their early and accurate detection, thus improving the sanitary status of cultivated plants in the near future.