Past and future evolution of Abies alba forests in Europe - comparison of a dynamic vegetation model with palaeo data and observations

Information on how species distributions and ecosystem services are impacted by anthropogenic climate change is important for adaptation planning. Palaeo data suggest that Abies alba formed forests under significantly warmer-than-present conditions in Europe and might be a native substitute for widespread drought-sensitive temperate and boreal tree species such as beech (Fagus sylvatica) and spruce (Picea abies) under future global warming conditions. Here, we combine pollen and macrofossil data, modern observations, and results from transient simulations with the LPX-Bern dynamic global vegetation model to assess past and future distributions of A. alba in Europe. LPX-Bern is forced with climate anomalies from a run over the past 21 000 years with the Community Earth System Model, modern climatology, and with 21st-century multimodel ensemble results for the high-emission RCP8.5 and the stringent mitigation RCP2.6 pathway. The simulated distribution for present climate encompasses the modern range of A. alba, with the model exceeding the present distribution in north-western and southern Europe. Mid-Holocene pollen data and model results agree for southern Europe, suggesting that at present, human impacts suppress the distribution in southern Europe. Pollen and model results both show range expansion starting during the Bølling–Allerød warm period, interrupted by the Younger Dryas cold, and resuming during the Holocene. The distribution of A. alba expands to the north-east in all future scenarios, whereas the potential (currently unrealized) range would be substantially reduced in southern Europe under RCP8.5. A. alba maintains its current range in central Europe despite competition by other thermophilous tree species. Our combined palaeoecological and model evidence suggest that A. alba may ensure important ecosystem services including stand and slope stability, infrastructure protection, and carbon sequestration under significantly warmer-than-present conditions in central Europe.

Rapid divergence in physiological and life-history traits between northern and southern populations of the British introduced neo-species, Senecio squalidus

Alien plants provide a unique opportunity to study evolution in novel environments, but relatively little is known about the extent to which they become locally adapted to different environments across their new range. Here, we compare northern and southern populations of the introduced species Senecio squalidus in Britain; S. squalidus has been in southern Britain for approximately 200 years and reached Scotland only about 50 years ago. We conducted common garden experiments at sites in the north and south of the species’ range in Britain. We also conducted glasshouse and growth chamber experiments to test the hypothesis that southern genotypes flower later, are more drought-tolerant, germinate and establish better at warmer temperatures, and are less sensitive to cold stress than their more northern counterparts. Results from the common garden experiments are largely consistent with the hypothesis of rapid adaptive divergence of populations of the species within the introduced range, with genotypes typically showing a home-site advantage. Results from the glasshouse and growth chamber experiments demonstrate adaptive divergence in ability to tolerate drought stress and high temperatures, as well as in phenology. In particular, southern genotypes were more tolerant of dry conditions and high temperatures and they flowered later than northern genotypes. Our results show that rapid local adaptation can occur in alien species, and they have implications for our understanding of the ecological genetics of range expansion of introduced weeds.

Differentiation of IncL and IncM Plasmids Associated with the Spread of Clinically Relevant Antimicrobial Resistance.

INTRODUCTION blaOXA-48, blaNDM-1 and blaCTX-M-3 are clinically relevant resistance genes, frequently associated with the broad-host range plasmids of the IncL/M group. The L and M plasmids belong to two compatible groups, which were incorrectly classified together by molecular methods. In order to understand their evolution, we fully sequenced four IncL/M plasmids, including the reference plasmids R471 and R69, the recently described blaOXA-48-carrying plasmid pKPN-El.Nr7 from a Klebsiella pneumoniae isolated in Bern (Switzerland), and the blaSHV-5 carrying plasmid p202c from a Salmonella enterica from Tirana (Albania). METHODS Sequencing was performed using 454 Junior Genome Sequencer (Roche). Annotation was performed using Sequin and Artemis software. Plasmid sequences were compared with 13 fully sequenced plasmids belonging to the IncL/M group available in GenBank. RESULTS Comparative analysis of plasmid genomes revealed two distinct genetic lineages, each containing one of the R471 (IncL) and R69 (IncM) reference plasmids. Conjugation experiments demonstrated that plasmids representative of the IncL and IncM groups were compatible with each other. The IncL group is constituted by the blaOXA-48-carrying plasmids and R471. The IncM group contains two sub-types of plasmids named IncM1 and IncM2 that are each incompatible. CONCLUSION This work re-defines the structure of the IncL and IncM families and ascribes a definitive designation to the fully sequenced IncL/M plasmids available in GenBank.

Generation of an hTERT-restricted, CMV-augmented oncolytic adenovirus capable of tumor-specific transgene expression: Development, characterization, and optimization

Current shortcomings in cancer therapy require the generation of new, broadly applicable, potent, targeted treatments. Here, an adenovirus is engineered to replicate specifically in cells with active human telomerase promotion using a modified hTERT promoter, fused to a CMV promoter element. The virus was also modified to contain a visible reporter transgene, GFP. The virus, Ad/hTC-GFP-E1 was characterized in vitro and demonstrated tumor specific activity both by dose and over time course experiments in a variety of cell lines. In vivo, Ad/hTC-GFP-E1 was affected at suppressing tumor growth and providing a survival benefit without causing any measurable toxicity. To increase the host range of the vector, the fiber region was modified to contain an RGD-motif. The vector, AdRGD/hTC-GFP-E1, was recharacterized in vitro, revealing heightened levels of infectivity and toxicity however maintaining a therapeutic window between cancer and normal cell toxicity. AdRGD/hTC-GFP-E1 was administered in vivo by limb perfusion and was observed to be tumor specific both in expression and replication. To further enhance the efficacy of viral vectors in lung delivery, asthma medications were investigated for their abilities to enhance transgene delivery and expression. A combination of bronchodilators, mast cell inhibitors, and mucolytic agents was devised which demonstrated fold increases in expression in immunocompetent mouse lungs as single agents and more homogenous, intense levels of expression when done in combination of all agents. To characterize the methods in which some cancers are resistant or may become resistant to oncolytic treatments, several small molecule inhibitors of metabolic pathways were applied in combination with oncolytic infection in vitro. SP600125 and PD 98059, respective JNK and ERK inhibitors, successfully suppressed oncolytic toxicity, however did not affect infectivity or transgene expression of Ad/hTC-GFP-E1. JNK and ERK inhibition did significantly suppress viral replication, however, as analyzed by lysate transfer and titration assays. In contrast, SB 203580, an inhibitor for p38, did not demonstrate any protective effects with infected cells. Flow cytometric analysis indicated a possible correlation with G1 arrest and suppressed viral production, however more compounds must be investigated to clarify this observation. ^

Clave para la identificación del segundo estadío larval de algunos trips comunes (Thysanoptera: Thripidae) : Mendoza, Argentina

Existen pocas publicaciones sobre claves de identificación de larvas de trips. Sin embargo, el tema es de interés básico para detectar el rango de hospedantes de los trips. Algunas especies son capaces de transmitir Tospovirus, agentes causales de la peste negra y otras enfermedades. El insecto adquiere el virus sólo como larva. Una clave de larvas será una herramienta útil para estudios epidemiológicos. La identificación de larvas es difícil porque tienen menos caracteres distintivos que los adultos; además, los individuos crecen constantemente. Las larvas obtenidas para este trabajo se recolectaron del campo sobre varias malezas y algunas plantas nativas y cultivadas. También se criaron en chauchas de poroto o polen y miel diluida a partir de adultos identificados. En este trabajo se presenta una descripción breve del segundo estadío larval de Frankliniella australis Morgan, F. gemina Bagnall, F. occidentalis Pergande, F. schultzei Trybom, F. valdiviana Sakimura et O'Neil y Thrips tabaci Lindeman y una clave para separar estas especies. Estación Experimental Agropecuaria INTA Mendoza.

Measurements of gastropods shell sizes from 23 fossil and extant long-lived lakes

Aim: To investigate shell size variation among gastropod faunas of fossil and recent long-lived European lakes and discuss potential underlying processes. Location: 23 long-lived lakes of the Miocene to Recent of Europe. Methods: Based on a dataset of 1412 species of both fossil and extant lacustrine gastropods, we assessed differences in shell size in terms of characteristics of the faunas (species richness, degree of endemism, differences in family composition) and the lakes (surface area, latitude and longitude of lake centroid, distance to closest neighbouring lake) using multiple and linear regression models. Because of a strong species-area relationship, we used resampling to determine whether any observed correlation is driven by that relationship. Results: The regression models indicated size range expansion rather than unidirectional increase or decrease as the dominant pattern of size evolution. The multiple regression models for size range and maximum and minimum size were statistically significant, while the model with mean size was not. Individual contributions and linear regressions indicated species richness and lake surface area as best predictors for size changes. Resampling analysis revealed no significant effects of species richness on the observed patterns. The correlations are comparable across families of different size classes, suggesting a general pattern. Main conclusions: Among the chosen variables, species richness and lake surface area are the most robust predictors of shell size in long-lived lake gastropods. Although the most outstanding and attractive examples for size evolution in lacustrine gastropods derive from lakes with extensive durations, shell size appears to be independent of the duration of the lake as well as longevity of a species. The analogue of long-lived lakes as 'evolutionary islands' does not hold for developments of shell size because different sets of parameters predict size changes.

AFLP data based on presence (1) and absence (0) of AFLP loci (A4-F171) in Gonyostomum semen

Many recent studies have found genetically differentiated populations in microorganisms despite potentially high dispersal. We designed a study to specifically examine the importance of physical dispersal barriers, i.e. geographic distance and lack of hydrological connectivity, in restricting gene flow and enhancing divergence in limnic microorganisms. We focused on the nuisance microalga Gonyostomum semen, which has recently expanded in northern Europe and differentiated into genetically distinct populations. Gonyostomum semen was sampled from six lakes distributed in two adjacent watersheds, which thereby comprised, both connected and non-connected lakes. The individual isolates were genotyped by Amplified Fragment Length Polymorphism. Several lake populations were differentiated from each other, but connectivity within watersheds could not explain the observed population genetic pattern. However, isolation by distance was moderate and might limit the gene flow among distant populations. In addition, we found low, but significant linkage disequilibrium, which indicates regular sexual recombination in this species, despite its high degree of asexual reproduction. Therefore, we conclude that the genetic properties of microalgae with occasional sexual reproduction essentially mirror regularly recombining species. Furthermore, the data indicated bottlenecks supporting the hypothesized recent range expansion of this species.

Using the critical salinity (Scrit) concept to predict invasion potential of the anemone Diadumene lineata in the Baltic Sea

It is widely assumed that the ability of an introduced species to acclimate to local environmental conditions determines its invasion success. The sea anemone Diadumene lineata is a cosmopolitan invader and shows extreme physiological tolerances. It was recently discovered in Kiel Fjord (Western Baltic Sea), although the brackish conditions in this area are physiologically challenging for most marine organisms. This study investigated salinity tolerance in D. lineata specimens from Kiel Fjord in order to assess potential geographical range expansion of the species in the Baltic Sea. In laboratory growth assays, we quantified biomass change and asexual reproduction rates under various salinity regimes (34: North Sea, 24: Kattegat, 14: Kiel Fjord, 7: Baltic Proper). Furthermore, we used 1H-NMR-based metabolomics to analyse intracellular osmolyte dynamics. Within 4 weeks D. lineata exhibited a 5-fold population growth through asexual reproduction at high salinities (34 and 24). Biomass increase under these conditions was significantly higher (69%) than at a salinity of 14. At a salinity of 7, anemones ceased to reproduce asexually, their biomass decreased and metabolic depression was observed. Five main intracellular osmolytes were identified to be regulated in response to salinity change, with osmolyte depletion at a salinity of 7. We postulate that depletion of intracellular osmolytes defines a critical salinity (Scrit) that determines loss of fitness. Our results indicate that D. lineata has the potential to invade the Kattegat and Skagerrak regions with salinity >10. However, salinities of the Baltic Proper (salinity <8) currently seem to constitute a physiological limit for the species.

Fusarium proliferatum isolated from garlic in Spain: Identification, toxigenic potential and pathogenicity on related Allium species

Fusarium proliferatum has been reported on garlic in the Northwest USA, Spain and Serbia, causing water-soaked tan-colored lesions on cloves. In this work, Fusarium proliferatum was isolated from 300 symptomatic garlic bulbs. Morphological identification of Fusarium was confirmed using species-specific PCR assays and EF-1α sequencing. Confirmation of pathogenicity was conducted with eighteen isolates. Six randomly selected F. proliferatum isolates from garlic were tested for specific pathogenicity and screened for fusaric acid production. Additionally, pathogenicity of each F. proliferatum isolate was tested on healthy seedlings of onion (Allium cepa), leek (A. porrum), scallions (A. fistulosum), chives (A. schoenoprasum) and garlic (A. sativum). A disease severity index (DSI) was calculated as the mean severity on three plants of each species with four test replicates. Symptoms on onion and garlic plants were observed three weeks after inoculation. All isolates tested produced symptoms on all varieties inoculated. Inoculation of F. proliferatum isolates from diseased garlic onto other Allium species provided new information on host range and pathogenicity. The results demonstrated differences in susceptibility with respect to host species and cultivar. The F. proliferatum isolates tested all produced fusaric acid (FA); correlations between FA production and isolate pathogenicity are discussed. Additionally, all isolates showed the presence of the FUM1 gene suggesting the ability of Spanish isolates to produce fumonisins.

The role of secretion systems and small molecules in soft-rot enterobacteriaceae pathogenicity

Soft-rot Enterobacteriaceae (SRE), which belong to the genera Pectobacterium and Dickeya, consist mainly of broad host-range pathogens that cause wilt, rot, and blackleg diseases on a wide range of plants. They are found in plants, insects, soil, and water in agricultural regions worldwide. SRE encode all six known protein secretion systems present in gram-negative bacteria, and these systems are involved in attacking host plants and competing bacteria. They also produce and detect multiple types of small molecules to coordinate pathogenesis, modify the plant environment, attack competing microbes, and perhaps to attract insect vectors. This review integrates new information about the role protein secretion and detection and production of ions and small molecules play in soft-rot pathogenicity.

Ecological and genetic determinants of Pepino mosaic virus emergence

Virus emergence is a complex phenomenon, which generally involves spread to a new host from a wild host, followed by adaptation to the new host. Although viruses account for the largest fraction of emerging crop pathogens, knowledge about their emergence is incomplete. We address here the question of whether Pepino mosaic virus (PepMV) emergence as a major tomato pathogen worldwide could have involved spread from wild to cultivated plant species and host adaptation. For this, we surveyed natural populations of wild tomatoes in southern Peru for PepMV infection. PepMV incidence, genetic variation, population structure, and accumulation in various hosts were analyzed. PepMV incidence in wild tomatoes was high, and a strain not yet reported in domestic tomato was characterized. This strain had a wide host range within the Solanaceae, multiplying efficiently in most assayed Solanum species and being adapted to wild tomato hosts. Conversely, PepMV isolates from tomato crops showed evidence of adaptation to domestic tomato, possibly traded against adaptation to wild tomatoes. Phylogenetic reconstructions indicated that the most probable ancestral sequence came from a wild Solanum species. A high incidence of PepMV in wild tomato relatives would favor virus spread to crops and its efficient multiplication in different Solanum species, including tomato, allowing its establishment as an epidemic pathogen. Later, adaptation to tomato, traded off against adaptation to other Solanum species, would isolate tomato populations from those in other hosts.

Native bradyrhizobial symbionts of Lupinus mariae-josephae, a unique endemism thriving in alkaline soils in Eastern Spain

Lupinus mariae-josephae (Lmj) es una especie de lupino endémica de una pequeña y específica área de Comunidad Valenciana (Este de España), donde prospera en suelos alcalinoscalcáreos, un hábitat singular para los altramuces, que crecen preferentemente en suelos ácidos o neutros. Esto hace de Lmj una especie de lupino única. Cuando se inició este trabajo, la extensión conocida de este endemismo abarcaba unos 700 kilómetros cuadrados, confinados en la provincia de Valencia. En esta área, Lmj prospera en pequeñas poblaciones aisladas que contienen un número reducido de plantas por lo que se la consideró una especie en peligro de extinción. Todos los esfuerzos, utilizando estrategias clásicas dirigidas a ampliar el área de crecimiento de Lmj y garantizar su conservación, han tenido un éxito limitado. El trabajo que se presenta está dirigido a mejorar el conocimiento de la ecología de Lmj, en particular la interacción simbiótica que establece con bacterias del suelo denominadas rizobios y se centra en la caracterización fenotípica, filogenética y genómica de esos rizobios. También se investiga la posible contribución de la simbiosis en mejorar la conservación de Lmj. Para este fin, se han estudiado diferentes aspectos que se describen a continuación. El primero objetivo se centró en aislar y estudiar de la diversidad genética de las bacterias endosimbióticas de Lmj. . Se realizó un análisis filogenético de genes esenciales que mostró que las cepas de Lmj pertenecen al género Bradyrhizobium y que presentan una gran diversidad con características fenotípicas y simbióticas diferentes de cepas de Bradyrhizobium que nodulan otras especies de lupinos nativos de España (cepas ISLU). Las cepas estudiadas se dividieron en dos grupos (Clado I y Clado II). El Clado I, incluye a las cepas Lmj, definiendo un nuevo linaje, filogenéticamente relacionado con otras especies de Bradyrhizobium, como B. jicamae y B. elkanii. El Clado II contiene cepas ISLU relacionadas con cepas de B. canariense y B. japonicum que establecen simbiosis con lupinos de suelos ácidos. Otro análisis filogenético basado en genes simbióticos, distribuyó las cepas de Lmj en sólo dos grupos diferentes. La singularidad y gran diversidad de estas cepas en una pequeña área geográfica, hacen de este, un atractivo sistema para el estudio de la evolución y adaptación de las bacterias simbióticas a su respectiva planta huésped. Adicionalmente, se estudio la presencia de bacterias capaces de nodular Lmj en suelos básicos de Chiapas, México. Sorprendentemente, estos suelos contienen bacterias capaces establecer interacciones simbióticas eficientes con Lmj en ensayos de invernadero. A continuación se investigó la taxonomía de los endosimbiontes de Lmj analizando la secuencia de cuatro genes esenciales (16S rRNA, recA, glnII y atpD) y el promedio de identidad de nucleótidos de genomas completos de algunas cepas representativas de la diversidad (ANIm). Se identificaron nuevas especies de Bradyrhizobium dentro del Clado I y se definió una de ellas: 'Bradyrhizobium valentinum' sp. nov (cepa tipo LmjM3T = CECT 8364T, LMG 2761T). También se abordó cómo conservar Lmj en su hábitat natural mediante inoculación con alguna de las cepas aisladas. Se demostró la ausencia de bacterias capaces de nodular Lmj en suelos rojos alcalinos o ‘‘terra rossa’’ de la Península Ibérica y Baleares. Dos cepas, altamente eficientes en cuanto a la fijación de nitrógeno, LmjC y LmjM3T, fueron seleccionadas para ser empleadas como inoculantes. Dos experimentos de campo llevados a cabo en años consecutivos en áreas con características edafoclimáticas similares a las que presentan las poblaciones de Lmj, lograron la reproducción exitosa de la planta. Se concluyó que un ciclo reproductivo exitoso de Lmj es absolutamente dependiente de la inoculación con sus simbiontes naturales y que la simbiosis debe ser considerada un factor esencial en estrategias de conservación de leguminosas en peligro. La obtención de varias secuencias genómicas de cepas aisladas de Lmj y de otras cepas de Bradyrhizobium reveló una alta similitud entre los genomas de las cepas del Clado I, y permitió la identificación de cinco posibles nuevas especies. Además, se estudiaron tres agrupaciones de genes relacionados con la simbiosis (nod, nif y fix) definiendo un nuevo linaje para las cepas de Lmj, diferente del symbiovar “genistearum” de B. canariense y B. japonicum. La baja diversidad encontrada en el análisis filogenético de los genes simbióticos contrasta con la gran diversidad asociada a genes esenciales. La presencia de plásmidos en cepas del género Bradyrhizobium ha sido descrita en muy pocas ocasiones, sin embargo el análisis de la secuencia genómica de la cepa ISLU101, aislada de Lupinus angustifolius, reveló la presencia de un origen de replicación extracromosómico homólogo al operón repABC, presente en el plásmido de Bradyrhizobium sp BTAi1. Gracias a esta secuencia se identificaron genes homólogos en 19 de 72 cepas ISLU. Filogenéticamente, las secuencias de repABC se agruparon en un grupo monofilético con las de pBTAi1 y separadas de los rizobios de crecimiento rápido. Finalmente, se identificaron sistemas de secreción de proteínas de tipo III (T3SS) en nueve genomas de cepas de Lmj. Los T3SS pueden inyectar proteínas efectoras al interior de células vegetales. Su presencia en rizobios se ha relacionado con la gama de hospedador que pueden nodular y puede tener un efecto beneficioso, neutro o perjudicial en la simbiosis. Los T3SS de las cepas de Lmj codifican para una proteína efectora similar a NopE, un efector dependiente de T3SS descrito en B. diazoefficiens USDA 110T. La proteína NopE de la cepa LmjC se ha caracterizado bioquímicamente. ABSTRACT Lupinus mariae-josephae (Lmj) is a lupine species endemic of a unique small area in Valencia region (Eastern Spain) where the lupine plants thrive in alkaline-limed soils, which preferentially grow in acid or neutral soils. This is the type of soils native lupines of Spain. When this work was initiated, the extension of the endemic area of Lmj was of about 700 squared kilometers confined to the Valencia province. In this area, Lmj thrives in small, isolated patches containing a reduced number of plants, and points to an endemism that can easily became endangered or extinct. Consequently, the Valencia Community authorities gave a ‘‘microreserve” status for conservation of the species. All efforts, using classical strategies directed to extend the area of Lmj growth and ensure its conservation have been so far unsuccessful. The work presented here is directed to improve our knowledge of Lmj ecology and it is centered in the characterization of the rhizobial symbiosis by phenotypic, phylogenetic and genomic analysis as well as in investigate the potential contribution of the symbiosis to improve its conservation. To this end, five different topics have been studied, and results are briefly described here. Extensive details can be followed en the attached, published articles. The first topic deals with the indigenous rhizobial symbionts of the Lmj endemism, and its genetic diversity was investigated. The Lmj root symbionts belong to the Bradyrhizobium genus, and phylogenetic analysis based on core genes identified a large diversity of Bradyrhizobium strains with phenotypic and symbiotic characteristics different from rhizobia nodulating other Lupinus spp. native of Spain. The strains were split in two clades. Clade II contained strains close to classical B. canariense and B. japonicum lineages that establish symbioses with lupines in acid soils of the Mediterranean area. Clade I included Lmj strains that define a new lineage, close to other Bradyrhizobium species as B. jicamae and B. elkanii. The phylogenetic analysis based on symbiotic genes identified only two distinct clusters. The singularity and large diversity of these strains in such a small geographical area makes this an attractive system for studying the evolution and adaptation of the rhizobial symbiont to the plant host. Additionally, the presence of bacteria able to nodulate Lmj in basic soils from Chiapas, Mexico was investigated. Surprisingly, these soils contain bacteria able to effectively nodulate and fix nitrogen with Lmj plants in greenhouse assays. In the second topic, the taxonomic status of the endosymbiotic bacteria of Lmj from Valencia endemism and Chiapas was investigated. Results from phylogenetic analysis of core genes and Average Nucleotide Identity (ANIm) using draft genomic sequences identified new Bradyrhizobium species within strains of Clade I of Lmj endosymbiotic bacteria. Only one of these potentially new species has been defined, meanwhile the others are under process of characterization. The name ‘Bradyrhizobium valentinum’ sp. nov. was proposed for the defined species (type strain LmjM3T= CECT 8364T, LMG 2761T). The third topic was directed to conservation of endangered Lmj in its natural habitat. The relevant conclusion of this experimentation is that the symbiosis should be considered as a relevant factor in the conservation strategies for endangered legumes. First, we showed absence of bacteria able to nodulate Lmj in all the inspected ‘‘terra rossa’’ or alkaline red soils of the Iberian Peninsula and Balearic Islands. Then, two efficient nitrogen fixing strains with Lmj plants, LmjC and LmjM3T, were selected as inoculum for seed coating. Two planting experiments were carried out in consecutive years under natural conditions in areas with edapho-climatic characteristics identical to those sustaining natural Lmj populations, and successful reproduction of the plant was achieved. The relevant conclusion from these assays was that the successful reproductive cycle was absolutely dependent on seedling inoculation with effective bradyrhizobia The forth topic deep into the analysis of the genomic of Lmj representative strains. To this end, draft genomic sequences of selected Lmj strains and type strains of Bradyrhizobium spp. were assembled. The comparison analysis of the draft genomic sequences of Lmj strains and related Bradyrhizobium species grouped in Clade I, revealed a high genomic homology among them, and allowed the definition of five potentially new species of Lmj nodulating bacteria. Also, based on the available draft genomic sequences, only three clusters of nod, fix and nif genes from Lmj strains were identified and showed to define a new symbiotic lineage, distant from that of B. canariense and B. japonicum bv. genistearum. The low diversity exhibited by the phylogenetic analysis of symbiotic genes contrast with the large diversity of strains as regards the housekeeping genes analyzed. Besides, the genomic analysis of a Lupinus angustifolius strain ISLU101, revealed the presence of an extrachromosomal replication origin homologous to repABC cluster from plasmid present in Bradyrhizobium spp BTAi1. This repABC cluster gene sequence allowed the identification of extrachromosomic replication origin in 19 out of 72 Bradyrhizobium strains from Lupinus spp., a highly significant result since the absence of plasmids in the Bradyrhizobium genus was traditionally assumed. The repABC gene sequences of these strains grouped them in a unique monophyletic group, related to B. sp. BTAi1 plasmid, but differentiated from the repABC gene cluster of plasmids in fast growing rhizobium strains. The last topic was focused on characterization of type III secreted effectors present in Lmj endosymbiotic bacteria. Type III secretion systems (T3SS) are specialized protein export machineries which can deliver effector proteins into plant cells. The presence of T3SS in rhizobia has frequently been related to the symbiotic nodulation host-range and may have a beneficial or detrimental effect on the symbiosis with legumes. In this context, the presence of T3SS in genomes of nine Lmj strains was investigated, and it was shown the presence of clusters encoding NopE type III-secreted protein similar to the NopE1 and NopE2 of B. diazoefficiens USDA 110T. The putative NopE protein of LmjC strain is at present being characterized regarding its structure and function.

Mecanismos implicados en las etapas iniciales de infección en la cancrosis de los cítricos provocada por Xanthomonas citri subsp. citri

La cancrosis o chancro bacteriano de los cítricos (CBC) causada por Xanthomonas citri subsp. citri (Xcc) y X. fuscans subsp. aurantifolii, afecta a un gran número de especies dentro de la familia de las rutáceas, especialmente cítricos. Esta enfermedad produce graves pérdidas económicas allí donde está presente, principalmente porque la comercialización de cítricos desde las zonas afectadas hacía zonas libres de cancrosis, está sujeta a fuertes medidas cuarentenarias. La cancrosis se encuentra distribuida a nivel mundial pero no se ha localizado ni en la Unión Europea ni en ningún área del Mediterráneo. Se han descrito tres tipos de cancrosis en función de la gama de huésped y de las características fenotípicas y genotípicas de las bacterias que las producen. La más extendida es la cancrosis tipo A producida por Xcc, dentro de la cual se distinguen los subtipos Aw y A*, originarios de Florida y Sudeste Asiático, respectivamente, que de forma natural solo son capaces de producir enfermedad en lima mejicana. En este trabajo se presentan estudios sobre mecanismos implicados en las primeras etapas de la infección, como la quimiotaxis y formación de biopelículas, en la cancrosis de los cítricos. La quimiotaxis es el proceso por el cual las bacterias se dirigen hacia zonas favorables para su supervivencia y desarrollo. Los perfiles quimiotácticos obtenidos frente a distintas fuentes de carbono, así como los estudios en relación al contenido de proteínas aceptoras de grupos metilo (MCPs), permitieron agrupar a las cepas de Xanthomonas estudiadas en este trabajo, de acuerdo a la enfermedad producida y a su gama de huésped. Todas las cepas mostraron quimiotaxis positiva frente a extractos de hoja y apoplasto de diferentes especies, sin embargo, Xcc 306, X. alfalfae subsp. citrumelonis (Xac) y X. campestris pv. campestris (Xc) manifestaron respuestas más específicas frente a extractos de apoplasto de hojas de naranjo dulce, lima y col china, respectivamente. Dicho resultado nos permite asociar el mecanismo de quimiotaxis con la capacidad de las cepas de Xanthomonas para colonizar estos huéspedes de forma específica. Las cepas estudiadas fueron capaces de realizar movimiento tipo swimming, twitching y sliding en distintos medios, siendo el movimiento swimming el único en el que se encontraron diferencias entre las cepas de Xcc con distinta gama de huésped. En este trabajo se ha estudiado además la formación de biopelículas en superficies bióticas y abióticas, un mecanismo importante tanto para la supervivencia en superficie vegetal como para el desarrollo de la infección. Las cepas de Xanthomonas estudiadas fueron capaces de formar biopelículas in vitro, siendo mayor en un medio que simula el apoplasto y que contiene una baja concentración de nutrientes en comparación con medios que contenían alta concentración de nutrientes. La formación de biopelículas en superficie vegetal se encontró relacionada, en las cepas patógenas de cítricos, con la capacidad para infectar un tejido o huésped determinado. Se han caracterizado algunos de los componentes de la matriz extracelular producida por Xcc, que compone hasta un 90% de las bipoelículas. Entre ellos destaca el ADN extracelular, que tiene un papel como adhesina en las primeras etapas de formación de biopelículas y estructural en biopelículas maduras. Además, se han identificado el pilus tipo IV como componente importante en las biopelículas, que también participa en motilidad. Finalmente, se han realizado estudios sobre la expresión de genes implicados en motilidad bacteriana y formación de biopelículas que han confirmado las diferencias existentes entre cepas de Xcc de amplia y limitada gama de huésped, así como el papel que juegan elementos como el pilus tipo IV o el flagelo en estos procesos. ABSTRACT Xanthomonas citri subsp. citri (Xcc) and X. fuscans subsp. aurantifolii are the causal agents of Citrus Bacterial Canker (CBC) which is one of the most important citrus diseases. CBC affects all Citrus species as well as other species from Rutaceae family. CBC produces strong economic losses; furthermore the commercialization of plants and fruits is restricted from infested to citrus canker free areas. The disease is worldwide distributed in tropical and subtropical areas, however it is not present in the European Union. Three types of CBC have been described according to the host range and phenotypic and genotypic characteristics. CBC type A caused by Xcc is he widest distributed. Within CBC A type two subtypes Aw and A* were described from Florida and Iran respectively, both infecting only Mexican lime. Herein mechanisms connected to early events in the citrus bacterial canker disease such as chemotaxis and biofilm formation, were studied. Chemotaxis allows bacteria to move towards the more suitable environments for its survival, host colonization and infection. Studies performed on citrus pathogenic Xanthomonas and X. campestris pv. campestris (Xc), a crucifer pathogen, have shown different chemotactic profiles towards carbon compound as well as different MCPs profile, which clustered strains according to host range and disease caused. Every strain showed positive chemotaxis toward leaf extracts and apoplastic fluids from sweet orange, Mexican lime and Chinese cabbage leaves. However, a more specific response was found for strains Xcc 306, X. alfalfae subsp. citrumelonis and Xc towards sweet orange, Mexican lime and Chinese cabbage apoplastic fluids, respectively. These results relate chemotaxis with the higher ability of those strains to specifically colonize their proper host. Xanthomonas strains studied were able to perform swimming, sliding and twitching motilities. The ability to swim was variable among CBC strains and seemed related to host range. Biofilm formation is an important virulence factor for Xcc because it allows a better survival onto the plant surface as well as facilitates the infection process. The studied Xanthomonas strains were able to form biofilm in vitro, on both nutrient rich and apoplast mimicking media, furthermore the biofilm formation by all the strains was higher in the apoplast mimicking media. The ability to form biofilm in planta by Xcc and Xac strains was dependent of the host and the tissue colonized. The wide host range CBC strain was able to form biofilm onto several citrus leaves and fruits, however the limited host range CBC strain produced biofilm solely onto Mexican lime leaves and fruits. Furthermore Xac strain, which solely infects leaves of young plants, was not able to develop biofilms on fruits. Some components of the extracellular matrix produced by Xcc strains have been characterized. Extracellular DNA acted as an adhesin at the very early stages of biofilm formation and as structural component of mature biofilm for citrus pathogenic Xanthomonas. Furthermore type IV pilus has been identified as a component of the extracellular matrix in biofilm and motility. Transcriptional studies of genes related with biofilm formation and motility have confirmed the differential behavior found among wide and limited host range CBC strains as well as the role of type IV pili and flagellum on those processes.