Enzyme activities of digestive and metabolic enzymes of Calanus finmarchicus from Maria S. Merian cruise MSM26 in spring 2013

The present study aimed to contribute to the knowledge on the intraspecific variations of enzyme activities in populations of Calanus finmarchicus from different longitudes across the North Atlantic Ocean and their relation to changing environmental conditions. C. finmarchicus was sampled across the North Atlantic in basins with decreasing temperature regimes from east to west (Iceland Basin, Irminger Basin and Labrador Basin) in late March/early April 2013. Potential maximum enzyme activities of digestive (proteinases and lipases/esterases) and metabolic (citrate synthase) enzymes of copepods from all sampling stations were analysed and thermal profiles (5-50°C) of enzyme activities were determined. In order to investigate its acclimation potential, C. finmarchicus were acclimated to 4°C and 15°C for two weeks and thermal profiles of enzyme activities were compared afterwards.

(Supplementary Table 1) Proportions of methane (C1) to ethane (C2) and the stable isotope composition of methane (d13CCH4) of gas and oil collected by gas bubble sampler during cruise M 114 in the southern Gulf of Mexico

Hydrocarbon seepage is a widespread process at the continental margins of the Gulf of Mexico. We used a multidisciplinary approach, including multibeam mapping and visual seafloor observations with different underwater vehicles to study the extent and character of complex hydrocarbon seepage in the Bay of Campeche, southern Gulf of Mexico. Our observations showed that seafloor asphalt deposits previously only known from the Chapopote Knoll also occur at numerous other knolls and ridges in water depths from 1230 to 3150 m. In particular the deeper sites (Chapopopte and Mictlan knolls) were characterized by asphalt deposits accompanied by extrusion of liquid oil in form of whips or sheets, and in some places (Tsanyao Yang, Mictlan, and Chapopote knolls) by gas emission and the presence of gas hydrates in addition. Molecular and stable carbon isotopic compositions of gaseous hydrocarbons suggest their primarily thermogenic origin. Relatively fresh asphalt structures were settled by chemosynthetic communities including bacterial mats and vestimentiferan tube worms, whereas older flows appeared largely inert and devoid of corals and anemones at the deep sites. The gas hydrates at Tsanyao Yang and Mictlan Knolls were covered by a 5-to-10 cm-thick reaction zone composed of authigenic carbonates, detritus, and microbial mats, and were densely colonized by 1-2 m-long tube worms, bivalves, snails, and shrimps. This study increased knowledge on the occurrences and dimensions of asphalt fields and associated gas hydrates at the Campeche Knolls. The extent of all discovered seepage structure areas indicates that emission of complex hydrocarbons is a widespread, thus important feature of the southern Gulf of Mexico.

(Table 3) Carbon and oxygen isotopic data of carbonates from Core SLR 37-1, HYC 25-1 and an aragonite crust from station DR 35-1

An area of massive barite precipitations was studied at a tectonic horst in 1500 m water depth in the Derugin Basin, Sea of Okhotsk. Seafloor observations and dredge samples showed irregular, block- to column-shaped barite build-ups up to 10 m high which were scattered over the seafloor along an observation track 3.5 km long. High methane concentrations in the water column show that methane expulsion and probably carbonate precipitation is a recently active process. Small fields of chemoautotrophic clams (Calyptogena sp., Acharax sp.) at the seafloor provide additional evidence for active fluid venting. The white to yellow barites show a very porous and often layered internal fabric, and are typically covered by dark-brown Mn-rich sediment; electron microprobe spectroscopy measurements of barite sub-samples show a Ba substitution of up to 10.5 mol% of Sr. Rare idiomorphic pyrite crystals (1%) in the barite fabric imply the presence of H2S. This was confirmed by clusters of living chemoautotrophic tube worms (1 mm in diameter) found in pores and channels within the barite. Microscopic examination showed that micritic aragonite and Mg-calcite aggregates or crusts are common authigenic precipitations within the barite fabric. Equivalent micritic carbonates and barite carbonate cemented worm tubes were recovered from sediment cores taken in the vicinity of the barite build-up area. Negative ?13C values of these carbonates (>?43.5? PDB) indicate methane as major carbon source; ?18O values between 4.04 and 5.88? PDB correspond to formation temperatures, which are certainly below 5°C. One core also contained shells of Calyptogena sp. at different core depths with 14C-ages ranging from 20 680 to >49 080 yr. Pore water analyses revealed that fluids also contain high amounts of Ba; they also show decreasing SO42- concentrations and a parallel increase of H2S with depth. Additionally, S and O isotope data of barite sulfate (?34S: 21.0-38.6? CDT; ?18O: 9.0-17.6? SMOW) strongly point to biological sulfate reduction processes. The isotope ranges of both S and O can be exclusively explained as the result of a mixture of residual sulfate after a biological sulfate reduction and isotopic fractionation with 'normal' seawater sulfate. While massive barite deposits are commonly assumed to be of hydrothermal origin, the assemblage of cheomautotrophic clams, methane-derived carbonates, and non-thermally equilibrated barite sulfate strongly implies that these barites have formed at ambient bottom water temperatures and form the features of a Giant Cold Seep setting that has been active for at least 49 000 yr.

Environmental assay on the effect of poultry manure application on soil organisms in agroecosystems

This paper reports the effects produced on the organisms of the soil (plants, invertebrates and microorganisms), after the application of two types of poultry manure (sawdust and straw bed) on an agricultural land. The test was made using a terrestrial microcosm, Multi-Species Soil System (MS3) developed in INIA. There was no difference in the germination for any of the three species of plants considered in the study. The biomass was increased in the wheat (Triticum aestivum) coming from ground treated with both kinds of poultry manure. Oilseed rape (Brasica rapa) was not affected and regarding vetch (Vicia sativa) only straw poultry manure showed significant difference. For length only Vicia sativa was affected showing a reduction when straw was exposed to poultry manure. When the effect on invertebrates was studied, we observed a reduction in the number of worms during the test, especially from the ground control (13.7%), higher than in the ground with sawdust poultry manure (6.7%), whereas in the ground with straw poultry manure, there was no reduction. The biomass was affected and at the end of the test it was observed that while the reduction of worms in the ground control was about 48%, the number of those that were in the ground with sawdust poultry manure or straw poultry manure decreased by 41% and 22% respectively. Finally, the effects on microorganisms showed that the enzymatic activities: dehydrogenase (DH) and phosphatase and basal respiration rate increased at the beginning of the test, and the differences were statistically significant compared with the values of the control group. During the test, all these parameters decreased (except DH activities) but they were always higher than in the ground control. This is why it is possible to deduce that the contribution of poultry manure caused an improvement in the conditions of fertilization and also for the soil.

Podredumbre parda del melocotonero (Monilinia spp.) : detección, identificación de especies y contribución a la epidemiología de la enfermedad

Monilinia spp. (M. laxa, M. fructigena y M. fructicola) causa marchitez en brotes y flores, chancros en ramas y podredumbre de la fruta de hueso provocando pérdidas económicas importantes en años con climatología favorable para el desarrollo de la enfermedad, particularmente en variedades tardías de melocotonero y nectarino. En estos huéspedes en España, hasta el momento, la especie predominante es M. laxa y, en menor proporción, M. fructigena. La reciente introducción en Europa de la especie de cuarentena M. fructicola hace necesaria una detección e identificación rápida de cada una de las especies. Además, hay diversos aspectos de la etiología y epidemiología de la enfermedad que no se conocen en las condiciones de cultivo españolas. En un primer objetivo de esta Tesis se ha abordado la detección e identificación de las especies de Monilinia spp. causantes de podredumbre parda. El estudio de las bases epidemiológicas para el control de la enfermedad constituye el fin del segundo objetivo. Para la detección del género Monilinia en material vegetal por PCR, diferenciándolo de otros hongos presentes en la superficie del melocotonero, se diseñaron una pareja de cebadores siguiendo un análisis del ADN ribosomal. La discriminación entre especies de Monilinia se consiguió utilizando marcadores SCAR (región amplificada de secuencia caracterizada), obtenidos después de un estudio de marcadores polimórficos de ADN amplificados al azar (RAPDs). También fue diseñado un control interno de amplificación (CI) basado en la utilización de un plásmido con secuencias de los cebadores diferenciadores del género, para ser utilizado en el protocolo de diagnóstico de la podredumbre parda con el fin de reconocer falsos negativos debidos a la inhibición de PCR por componentes celulares del material vegetal. Se disponía de un kit comercial que permitía distinguir Monilinia de otros géneros y M. fructicola del resto de especies mediante anticuerpos monoclonales utilizando la técnica DAS-ELISA. En esta Tesis se probaron diferentes fuentes de material como micelio ó conidias procedentes de cultivos en APD, o el micelio de la superficie de frutas o de momias frescas, como formas de antígeno. Los resultados obtenidos con ELISA se compararon con la identificación por métodos morfológico-culturales y por PCR con los cebadores desarrollados en esta Tesis. Los resultados demostraron la posibilidad de una detección temprana en frutas frescas por este método, realzando las posibilidades de una diagnosis temprana para una prevención más eficaz de M. fructicola en fruta de hueso. El estudio epidemiológico de la enfermedad comenzó con la determinación de las principales fuentes de inóculo primario y su importancia relativa en melocotoneros y nectarinos del valle del Ebro. Para ello se muestrearon 9 huertos durante los años 2003 a 2005 recogiendo todas las momias, frutos abortados, gomas, chancros, y brotes necróticos en los árboles. También se recogieron brotes aparentemente sanos y muestras de material vegetal situados en el suelo. En estas muestras se determinó la presencia de Monilinia spp. Los resultados mostraron que la fuente principal de inóculo son las momias que se quedan en los árboles en las que la supervivencia del hongo tras el invierno es muy alta. También son fuentes de inóculo las momias del suelo y los brotes necróticos. De aquí se deriva que una recomendación importante para los agricultores es que deben eliminar este material de los huertos. Un aspecto no estudiado en melocotonero o nectarino en España es la posible relación que puede darse entre la incidencia de infecciones latentes en los frutos inmaduros a lo largo del cultivo y la incidencia de podredumbre en los frutos en el momento de la recolección y en postcosecha. Esta relación se había observado previamente en otros frutales de hueso infectados con M. fructicola en diversos países del mundo. Para estudiar esta relación se realizaron ensayos en cinco huertos comerciales de melocotonero y nectarino situados en el Valle del Ebro en cuatro estados fenológicos durante los años 2000-2002. No se observaron infecciones latentes en botón rosa, dándose la máxima incidencia en precosecha, aunque en algunos huertos se daba otro pico en el endurecimiento del embrión. La especie prevaleciente fue M. laxa. Se obtuvo una correlación positiva significativa entre la incidencia de infecciones latentes y la incidencia de podredumbre en postcosecha. Se desarrolló también un modelo de predicción de las infecciones latentes en función de la temperatura (T) y el periodo de humectación (W). Este modelo indicaba que T y W explicaban el 83% de la variación en la incidencia de infecciones latentes causadas por Monilinia spp. Por debajo de 8ºC no se predecían latentes, necesitándose más de 22h de W para predecir la ocurrencia de latentes con T = 8ºC, mientras que solo se necesitaban 5h de W a 25ºC. Se hicieron también ensayos en condiciones controladas para determinar la relación entre la incidencia de las infecciones latentes, las condiciones ambientales (T y W), la concentración de inóculo del patógeno (I) y el estado de desarrollo del huésped (S) y para validar el modelo de predicción desarrollado con los experimentos de campo. Estos ensayos se llevaron cabo con flores y frutos de nectarino procedentes de un huerto comercial en seis estados fenológicos en los años 2004 y 2005, demostrándose que la incidencia de podredumbre en postcosecha y de infecciones latentes estaba afectada por T, W, I y S. En los frutos se producían infecciones latentes cuando la T no era adecuada para el desarrollo de podredumbre. Una vez desarrollado el embrión eran necesarias más de 4-5h de W al día y un inóculo superior a 104 conidias ml-1 para que se desarrollase o podredumbre o infección latente. La ecuación del modelo obtenido con los datos de campo era capaz de predecir los datos observados en estos experimentos. Para evaluar el efecto del inóculo de Monilinia spp. en la incidencia de infecciones latentes y de podredumbre de frutos en postcosecha se hicieron 11 experimentos en huertos comerciales de melocotonero y nectarino del Valle del Ebro durante 2002 a 2005. Se observó una correlación positiva entre los números de conidias de Monilinia spp. en la superficie de los frutos y la incidencia de infecciones latentes De los estudios anteriores se deducen otras dos recomendaciones importantes para los agricultores: las estrategias de control deben tener en cuenta las infecciones latentes y estimar el riesgo potencial de las mismas basándose en la T y W. Deben tener también en cuenta la concentración de esporas de Monilinia spp. en la superficie de los frutos para disminuir el riesgo de podredumbre parda. El conocimiento de la estructura poblacional de los patógenos sienta las bases para establecer métodos más eficaces de manejo de las enfermedades. Por ello en esta Tesis se ha estudiado el grado de diversidad genética entre distintas poblaciones de M. laxa en diferentes localidades españolas utilizando 144 marcadores RAPDs (59 polimórficos y 85 monomórficos) y 21 aislados. El análisis de la estructura de la población reveló que la diversidad genética dentro de las subpoblaciones (huertos) (HS) representaba el 97% de la diversidad genética (HT), mientras que la diversidad genética entre subpoblaciones (DST) sólo representaba un 3% del total de esta diversidad. La magnitud relativa de la diferenciación génica entre subpoblaciones (GST) alcanzaba 0,032 y el número estimado de migrantes por generación (Nm) fue de 15,1. Los resultados obtenidos en los dendrogramas estaban de acuerdo con el análisis de diversidad génica. Las agrupaciones obtenidas eran independientes del huerto de procedencia, año o huésped. En la Tesis se discute la importancia relativa de las diferentes fuentes evolutivas en las poblaciones de M. laxa. Finalmente se realizó un muestreo en distintos huertos de melocotonero y nectarino del Valle del Ebro para determinar la existencia o no de aislados resistentes a los fungicidas del grupo de los benzimidazoles y las dicarboximidas, fungicidas utilizados habitualmente para el control de la podredumbre parda y con alto riesgo de desarrollar resistencia en las poblaciones patógenas. El análisis de 114 aislados de M. laxa con los fungicidas Benomilo (bencimidazol) (1Bg m.a ml-1), e Iprodiona (dicarboximida) (5Bg m.a ml-1), mostró que ninguno era resistente en las dosis ensayadas. Monilinia spp. (M. laxa, M. fructigena and M. fructicola) cause bud and flower wilt, canker in branches and stone fruit rot giving rise important economic losses in years with appropriate environmental conditions, it is particularly important in late varieties of peach and nectarine. Right now, M. laxa is the major species for peach and nectarine in Spain followed by M. fructigena, in a smaller proportion. The recent introduction of the quarantine organism M. fructicola in Europe makes detection and identification of each one of the species necessary. In addition, there are different aspects of disease etiology and epidemiology that are not well known in Spain conditions. The first goal of this Thesis was the detection and identification of Monilinia spp. causing brown rot. Study of the epidemiology basis for disease control was the second objective. A pair of primers for PCR detection was designed based on the ribosomal DNA sequence in order to detect Monilinia spp. in plant material and to discriminate it from other fungi colonizing peach tree surface. Discrimination among Monilinia spp. was successful by SCAR markers (Sequence Characterized Amplified Region), obtained after a random amplified polymorphic DNA markers (RAPDs) study. An internal control for the PCR (CI) based on the use of a mimic plasmid designed on the primers specific for Monilinia was constructed to be used in diagnosis protocol for brown rot in order to avoid false negatives due to the inhibition of PCR as consequence of remained plant material. A commercial kit based on DAS-ELISA and monoclonals antibodies was successfully tested to distinguish Monilinia from other fungus genera and M. fructicola from other Monilinia species. Different materials such as micelium or conidias from APD cultures, or micelium from fresh fruit surfaces or mummies were tested in this Thesis, as antigens. Results obtained by ELISA were compared to classical identification by morphologic methods and PCR with the primers developed in this Thesis. Results demonstrated the possibility of an early detection in fresh fruits by this method for a more effective prevention of M. fructicola in stone fruit. The epidemiology study of the disease started with the determination of the main sources of primary inoculum and its relative importance in peach trees and nectarines in the Ebro valley. Nine orchards were evaluated during years 2003 to 2005 collecting all mummies, aborted fruits, rubbers, cankers, and necrotic buds from the trees. Apparently healthy buds and plant material located in the ground were also collected. In these samples presence of Monilinia spp. was determined. Results showed that the main inoculum sources are mummies that stay in the trees and where fungus survival after the winter is very high. Mummies on the ground and the necrotics buds are also sources of inoculum. As consequence of this an important recommendation for the growers is the removal of this material of the orchards. An important issue not well studied in peach or nectarine in Spain is the possible relationship between the incidence of latent infections in the immature fruits and the incidence of fruit rot at harvesting and postharvesting. This relationship had been previously shown in other stone fruit trees infected with M. fructicola in different countries over the world. In order to study this relationship experiments were run in five commercial peach and nectarine orchards located in the Ebro Valley in four phenologic states from 2000 to 2002. Latent infections were not observed in pink button, the maxima incidence arise in preharvest, although in some orchards another increase occurred in the embryo hardening. The most prevalence species was M. laxa. A significant positive correlation between the incidence of latent infections and the incidence of rot in postharvest was obtained. A prediction model of the latent infections based on the temperature (T) and the wetness duration (W) was also developed. This model showed that T and W explained 83% of the variation in latent infection incidence caused by Monilinia spp. Below 8ºC latent infection was not predicted, more than 22h of W with T = 8ºC were needed to predict latent infection occurrence of, whereas at 25ºC just 5h of W were enough. Tests under controlled conditions were also made to determine the relationship among latent infections incidence, environmental conditions (T and W), inoculum concentration of the pathogen (I) and development state of the host (S) to validate the prediction model developed on the field experiments. These tests were made with flowers and fruits of nectarine coming from a commercial orchard, in six phenologic states in 2004 and 2005, showing that incidence of rot in postharvest and latent infections were affected by T, W, I and S. In fruits latent infections took place when the T was not suitable for rot development. Once developed the embryo, more than 4-5h of W per day and higher inoculums (104 conidia ml-1) were necessary for rot or latent infection development. The equation of the model obtained with the field data was able to predict the data observed in these experiments. In order to evaluate the effect of inoculum of Monilinia spp. in the incidence of latent infections and of rot of fruits in postharvest, 11 experiments in commercial orchards of peach and nectarine of the Ebro Valley were performed from 2002 to 2005. A positive correlation between the conidial numbers of Monilinia spp. in the surface of the fruits and the incidence of latent infections was observed. Based on those studies other two important recommendations for the agriculturists are deduced: control strategies must consider the latent infections and potential risk based on the T and W. Spores concentration of Monilinia spp. in the surface of fruits must be also taken in concern to reduce the brown rot risk. The knowledge of the population structure of the pathogens determines the bases to establish more effective methods of diseases handling. For that reason in this Thesis the degree of genetic diversity among different M. laxa populations has been studied in different Spanish locations using 144 RAPDs markers (59 polymorphic and 85 monomorphics) on 21 fungal isolates. The analysis of the structure of the population revealed that the genetic diversity within the subpopulations (orchards) (HS) represented 97% of the genetic diversity (HT), whereas the genetic diversity between subpopulations (DST) only represented a 3% of the total of this diversity. The relative magnitude of the genic differentiation between subpopulations (GST) reached 0.032 and the considered number of migrantes by generation (Nm) was of 15.1. The results obtained in dendrograms were in agreement with the analysis of genic diversity. The obtained groupings were independent of the orchard of origin, year or host. In the Thesis the relative importance of the different evolutionary sources in the populations from M. laxa is discussed. Finally a sampling of resistant isolates in different orchards from peach and nectarine of Ebro Valley was made to determine the existence of fungicide resistance of the group of benzimidazoles and the dicarboximidas, fungicides used habitually for the control of rot brown and with high risk of resistance developing in the pathogenic populations. The analysis of 114 isolated ones of M. laxa with the fungicides Benomilo (bencimidazol) (1Bg m.a ml-1), and Iprodiona (dicarboximida) (5Bg m.a ml-1), showed no resistant in the doses evaluated.

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.

A human Cds1-related kinase that functions downstream of ATM protein in the cellular response to DNA damage

Checkpoints maintain the order and fidelity of the eukaryotic cell cycle, and defects in checkpoints contribute to genetic instability and cancer. Much of our current understanding of checkpoints comes from genetic studies conducted in yeast. In the fission yeast Schizosaccharomyces pombe (Sp), SpRad3 is an essential component of both the DNA damage and DNA replication checkpoints. The SpChk1 and SpCds1 protein kinases function downstream of SpRad3. SpChk1 is an effector of the DNA damage checkpoint and, in the absence of SpCds1, serves an essential function in the DNA replication checkpoint. SpCds1 functions in the DNA replication checkpoint and in the S phase DNA damage checkpoint. Human homologs of both SpRad3 and SpChk1 but not SpCds1 have been identified. Here we report the identification of a human cDNA encoding a protein (designated HuCds1) that shares sequence, structural, and functional similarity to SpCds1. HuCds1 was modified by phosphorylation and activated in response to ionizing radiation. It was also modified in response to hydroxyurea treatment. Functional ATM protein was required for HuCds1 modification after ionizing radiation but not after hydroxyurea treatment. Like its fission yeast counterpart, human Cds1 phosphorylated Cdc25C to promote the binding of 14-3-3 proteins. These findings suggest that the checkpoint function of HuCds1 is conserved in yeast and mammals.