First report of Fusarium verticillioides causing stalk and root rot of sorghum in Spain

Podredumbre del tallo y la raíz del sorgo causada por Fusarium verticillioides en España

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.

The effects of storage duration, temperature and cultivar on the severity of garlic clove rot caused by Fusarium proliferatum.

Diseases that affect garlic during storage can lead to severe economic losses for farmers worldwide. One causal agent of clove rot is Fusarium proliferatum. Here, the progress of clove rot caused by F. proliferatum and its dependence on different storage conditions and cultivar type were studied. The effect of temperature on mycelial growth, conidial viability, and fungal survival during garlic commercial storage was documented. Samples of 50 bulbs from a randomized field trial with three different clonal generations for purple garlic (F3, F4 and F5) and the F4 clonal generation for white garlic were labeled and stored for two months (short-term storage). In addition, another sample of the F5 clonal generation of purple garlic was stored for 6 months after harvest (long-term storage). The presence of the pathogen and the percentage of symptomatic cloves were evaluated. A notable difference in the rot severity index (RSI) of different garlic varieties was observed. In all studied cases, clove rot increased with storage time at 20 ◦ C, and the white garlic variety had a higher index of rot severity after two months of storage. Additionally, there were clear differences between the growth rates of F. proliferatum isolates. Studies conducted on the temperature responses of the pathogen propagules showed that expo- sure for at least 20 min at 50 ◦ C was highly effective in significantly reducing the viability of fungal conidia. Pathogenicity studies showed that the fungus is pathogenic in all commercial varieties. However, there were significant differences in varietal susceptibility between Chinese and white garlic type cultivars (81.84 ± 16.44% and 87.5 ± 23.19% symptomatic cloves, respectively) and purple cultivars (49.06 ± 13.42% symptomatic cloves)

Seed Dispersal Spectrum of Woody Species in South Ecuadorian Dry Forests: Environmental Correlates and the Effect of Considering Species Abundance

This study examines the seed dispersal spectrum of the tropical dry forests of Southern Ecuador, in an effort to contribute to the knowledge of the complex dynamics of tropical dry forests. Seed dispersal spectrum was described for a total number of 160 species. Relationships of dispersal syndromes with plant growth form and climatic seasonality were explored. For a subset of 97 species, we determined whether dispersal spectrum changes when species abundance, in addition to species number, is taken into account. The same subset was used to relate dispersal syndromes with the environmental conditions. Zoochorous species dominated in the studied community. When considering the individual abundance of each species, however, anemochory was the prevalent dispersal syndrome. We found a significant difference in the frequency of dispersal syndromes among plant growth forms, with epizoochory only occurring in shrub species. The dispersal spectrum was dependent on climatic seasonality. The largest proportion of anemochorous species fructified during the dry season, while zoochorous diaspores dominated during the rainy season. A fourth-corner analysis indicated that the seed dispersal spectrum of Southern Ecuador dry forests is controlled by environmental conditions such as annual precipitation, annual temperature range or topography. Our results suggest that spatio-temporal changes in the environmental conditions may affect important ecological processes for dispersal. Thus, the predominance of one syndrome or another may depend on the spatial variation of environmental conditions.

Competition may explain the fine-scale spatial patterns and genetic structure of two co-occurring plant congeners

1. The spatial distribution of individual plants within a population and the population’s genetic structure are determined by several factors, like dispersal, reproduction mode or biotic interactions. The role of interspecific interactions in shaping the spatial genetic structure of plant populations remains largely unknown. 2. Species with a common evolutionary history are known to interact more closely with each other than unrelated species due to the greater number of traits they share. We hypothesize that plant interactions may shape the fine genetic structure of closely related congeners. 3. We used spatial statistics (georeferenced design) and molecular techniques (ISSR markers) to understand how two closely related congeners, Thymus vulgaris (widespread species) and T. loscosii (narrow endemic) interact at the local scale. Specific cover, number of individuals of both study species and several community attributes were measured in a 10 × 10 m plot. 4. Both species showed similar levels of genetic variation, but differed in their spatial genetic structure. Thymus vulgaris showed spatial aggregation but no spatial genetic structure, while T. loscosii showed spatial genetic structure (positive genetic autocorrelation) at short distances. The spatial pattern of T. vulgaris’ cover showed significant dissociation with that of T. loscosii. The same was true between the spatial patterns of the cover of T. vulgaris and the abundance of T. loscosii and between the abundance of each species. Most importantly, we found a correlation between the genetic structure of T. loscosii and the abundance of T. vulgaris: T. loscosii plants were genetically more similar when they were surrounded by a similar number of T. vulgaris plants. 5. Synthesis. Our results reveal spatially complex genetic structures of both congeners at small spatial scales. The negative association among the spatial patterns of the two species and the genetic structure found for T. loscosii in relation to the abundance of T. vulgaris indicate that competition between the two species may account for the presence of adapted ecotypes of T. loscosii to the abundance of a competing congeneric species. This suggests that the presence and abundance of close congeners can influence the genetic spatial structure of plant species at fine scales.

A method to incorporate the effect of taxonomic uncertainty on multivariate analyses of ecological data

Researchers in ecology commonly use multivariate analyses (e.g. redundancy analysis, canonical correspondence analysis, Mantel correlation, multivariate analysis of variance) to interpret patterns in biological data and relate these patterns to environmental predictors. There has been, however, little recognition of the errors associated with biological data and the influence that these may have on predictions derived from ecological hypotheses. We present a permutational method that assesses the effects of taxonomic uncertainty on the multivariate analyses typically used in the analysis of ecological data. The procedure is based on iterative randomizations that randomly re-assign non identified species in each site to any of the other species found in the remaining sites. After each re-assignment of species identities, the multivariate method at stake is run and a parameter of interest is calculated. Consequently, one can estimate a range of plausible values for the parameter of interest under different scenarios of re-assigned species identities. We demonstrate the use of our approach in the calculation of two parameters with an example involving tropical tree species from western Amazonia: 1) the Mantel correlation between compositional similarity and environmental distances between pairs of sites, and; 2) the variance explained by environmental predictors in redundancy analysis (RDA). We also investigated the effects of increasing taxonomic uncertainty (i.e. number of unidentified species), and the taxonomic resolution at which morphospecies are determined (genus-resolution, family-resolution, or fully undetermined species) on the uncertainty range of these parameters. To achieve this, we performed simulations on a tree dataset from southern Mexico by randomly selecting a portion of the species contained in the dataset and classifying them as unidentified at each level of decreasing taxonomic resolution. An analysis of covariance showed that both taxonomic uncertainty and resolution significantly influence the uncertainty range of the resulting parameters. Increasing taxonomic uncertainty expands our uncertainty of the parameters estimated both in the Mantel test and RDA. The effects of increasing taxonomic resolution, however, are not as evident. The method presented in this study improves the traditional approaches to study compositional change in ecological communities by accounting for some of the uncertainty inherent to biological data. We hope that this approach can be routinely used to estimate any parameter of interest obtained from compositional data tables when faced with taxonomic uncertainty.

Atlas y Libro Rojo de la Flora Vascular Amenazada de España. Manual de metodología del trabajo corológico y demográfico

El proyecto ''Atlas y Libro Rojo de la Flora Vascular Amenazada de España" (proyecto AFA) se ha desarrollado a iniciativa del Ministerio de Medio Ambiente, en el que han participado de forma coordinada más de 200 personas organizadas en una treintena de equipos de trabajo procedentes de universidades, centros de investigación y otras instituciones vinculadas a la conservación de plantas. Su objetivo principal es el inventariado basado en la cartografía, censo y catalogación de la flora vascular amenazada española. Este proyecto se encuentra enmarcado dentro en un extenso programa nacional de caracterización de la biodiversidad, denominado Inventario Nacional de Biodiversidad, que tiene como finalidad la creación y el mantenimiento a largo plazo de un inventario de la biodiversidad española, organizado en una serie de Atlas estructurados por grupos taxonómicos (http://www.mma.es/portal! secciones/biodiversidad / inventarios / inb/) . En el caso de la flora vascular, un total de 466 especies prioritarias, en su mayoría pertenecientes a las categorías "en peligro crítico" (CR) y "en peligro" (EN) se encuentran informatizadas en una base de datos del Ministerio de Medio Ambiente, I cuyos campos describen su corología en cuadrículas de 500 x 500 m, el tamaño de cada una de sus poblaciones, los factores de amenaza, el grado de protección territorial, las actuaciones emprendidas y las propuestas futuras de conservación. Una síntesis de dicha información fue publicada en 2003 (reeditada en 2004 y 2007) bajo el título ''Atlas y Libro Rojo de la Flora Vascular Amenazada de España" (Bañares el al., 2004). En un proceso continuo de ampliación se han sumado al proyecto otras series de 35 y 53 especies (mayoritariamente "vulnerables", VU), publicadas como adendas al Atlas y Libro Rojo en años sucesivos (Bañares el al., 2007, 2009). En el inicio de las labores organizativas del proyecto AFA, y con antelación a los trabajos de campo, se constituyó un grupo de trabajo con el objetivo de preparar un manual metodológico de obtención de datos aplicable a todos los taxones de flora vascular considerados y en todo el territorio. Este manual de metodología, que fue presentado a los equipos de trabajo en una reunión técnica celebrada en Miraflores de la Sierra (Madrid) en febrero de 2001 y que se publica con la presente edición, recopila las pautas dadas a los equipos de trabajo que participaron en la obtención de los datos de campo. Con la publicación de este Manual de Metodología aplicado en la ejecución del proyecto AFA se intenta lograr un doble objetivo: por un lado, divulgar la metodología empleada a un público más amplio al objeto de que pueda servir de base para la ejecución de otros estudios de la misma naturaleza en éste u otros entornos geográficos; en segundo lugar, dar máxima difusión a esta información para facilitar la posibilidad de que, en un futuro, cuando se emprendan acciones de naturaleza semejante sobre las plantas vasculares amenazadas de España, resulte posible comparar los resultados obtenidos en tal estudio con los publicados en el Atlas y Libro Rojo de la Flora Vascular Amenazada de España. La experiencia adquirida tras la aplicación de esta metodología a los más de 500 taxones estudiados durante estos años, más una serie de avances, fundamentalmente el acceso a ciertas herramientas como los Sistemas Globales de Navegación por Satélite (GNSS) (p.ej. GPS), los Sistemas de Información Geográfica (SIG), la fotografía digital y también el desarrollo de ciertas bases de datos fácilmente consultables, nos ha permitido ahora incluir un apartado adicional que recopila nuevas recomendaciones metodológicas a incorporar en futuros estudios de esta naturaleza.

Fine spatial pattern of an epiphytic lichen species is affected by habitat conditions in two forest types in the Iberian Mediterranean region

Persistence and abundance of species is determined by habitat availability and the ability to disperse and colonize habitats at contrasting spatial scales. Favourable habitat fragments are also heterogeneous in quality, providing differing opportunities for establishment and affecting the population dynamics of a species. Based on these principles, we suggest that the presence and abundance of epiphytes may reflect their dispersal ability, which is primarily determined by the spatial structure of host trees, but also by host quality. To our knowledge there has been no explicit test of the importance of host tree spatial pattern for epiphytes in Mediterranean forests. We hypothesized that performance and host occupancy in a favourable habitat depend on the spatial pattern of host trees, because this pattern affects the dispersal ability of each epiphyte and it also determines the availability of suitable sites for establishment. We tested this hypothesis using new point pattern analysis tools and generalized linear mixed models to investigate the spatial distribution and performance of the epiphytic lichen Lobaria pulmonaria, which inhabits two types of host trees (beeches and Iberian oaks). We tested the effects on L. pulmonaria distribution of tree size, spatial configuration, and host tree identity. We built a model including tree size, stand structure, and several neighbourhood predictors to understand the effect of host tree on L. pulmonaria. We also investigated the relative importance of spatial patterning on the presence and abundance of the species, independently of the host tree configuration. L. pulmonaria distribution was highly dependent on habitat quality for successful establishment, i.e., tree species identity, tree diameter, and several forest stand structure surrogates. For beech trees, tree diameter was the main factor influencing presence and cover of the lichen, although larger lichen-colonized trees were located close to focal trees, i.e., young trees. However, oak diameter was not an important factor, suggesting that bark roughness at all diameters favoured lichen establishment. Our results indicate that L. pulmonaria dispersal is not spatially restricted, but it is dependent on habitat quality. Furthermore, new spatial analysis tools suggested that L. pulmonaria cover exhibits a distinct pattern, although the spatial pattern of tree position and size was random.

Intra-annual patterns of tracheid size in the Mediterranean tree Juniperus thurifera as an indicator of seasonal water stress

Because climate can affect xylem cell anatomy, series of intra-annual cell anatomical features have the potential to retrospectively supply seasonal climatic information. In this study, we explored the ability to extract information about water stress conditions from tracheid features of the Mediterranean conifer Juniperus thurifera L. Tracheidograms of four climatic years from two drought-sensitive sites in Spain were compared to evaluate whether it is possible to link intra-annual cell size patterns to seasonal climatic conditions. Results indicated site-specific anatomical adjustment such as smaller and thicker tracheids at the dryer site but also showed a strong climatic imprint on the intra-annual pattern of tracheid size. Site differences in cell size reflected expected structural adjustments against cavitation failures. Differences between intra-annual patterns, however, indicated a response to seasonal changes in water availability whereby cells formed under drought conditions were smaller and thicker, and vice versa. This relationship was more manifest and stable at the dryer site

Juvenile–adult tree associations in a continental Mediterranean ecosystem: no evidence for sustained and general facilitation at increased aridity

Question: How do tree species identity, microhabitat and water availability affect inter- and intra-specific interactions between juvenile and adult woody plants? Location: Continental Mediterranean forests in Alto Tajo Natural Park, Guadalajara, Spain. Methods: A total of 2066 juveniles and adults of four co-occurring tree species were mapped in 17 plots. The frequency of juveniles at different microhabitats and water availability levels was analysed using log-linear models. We used nearest-neighbour contingency table analysis of spatial segregation and J-functions to describe the spatial patterns. Results: We found a complex spatial pattern that varied according to species identity and microhabitat. Recruitment was more frequent in gaps for Quercus ilex, while the other three species recruited preferentially under shrubs or trees depending on the water availability level. Juveniles were not spatially associated to conspecific adults, experiencing segregation from them inmany cases. Spatial associations, both positive and negative, were more common at higher water availability levels. Conclusions: Our results do not agree with expectations from the stressgradient hypothesis, suggesting that positive interactions do not increase in importance with increasing aridity in the study ecosystem. Regeneration patterns are species-specific and depend on microhabitat characteristics and dispersal strategies. In general, juveniles do not look for conspecific adult protection. This work contributes to the understanding of species co-existence, proving the importance of considering a multispecies approach at several plots to overcome limitations of simple pair-wise comparisons in a limited number of sites.

Estructura y funcionamiento de ecosistemas secos del Sur de Ecuador.

En la actualidad, los bosques secos neotropicales se encuentran restringidos a una pequeña fracción de su área de distribución histórica, debido principalmente a la acelerada pérdida de cobertura vegetal. Es por esta razón que son reconocidos como uno de los ecosistemas más amenazados del mundo. Durante las últimas décadas estos bosques han recibido mayor atención por parte de investigadores, y pese a que el esfuerzo dedicado al estudio y conocimiento de estos bosques no es comparable al de otros bosques de la región, especialmente los ligados a las pluviselvas húmedas, los trabajos llevados a cabo fundamentalmente por botánicos han permitido describir una buena parte de su diversidad florística en amplias regiones del neotrópico, constatar el elevado nivel de endemicidad de su flora y determinar algunos de los factores que determinan su estructura y funcionamiento. Sin embargo, aún existen regiones como la denominada Pacífico Ecuatorial donde la investigación ha sido escaza. El conocimiento de la diversidad biológica así como cuáles son los factores que controlan el funcionamiento y la estructuración de estos bosques resultan prioritarios para poder desarrollar acciones de conservación efectivas. El presente trabajo aporta a la comprensión del funcionamiento de dos sistemas secos tropicales de la región del Pacífico Ecuatorial el matorral seco y el bosque seco del sur del Ecuador. Se estableció una parcela de 9ha en bosque seco con el fin de evaluar asociaciones intraespecíficas e interespecíficas, se registró la diversidad y abundancia de todas las especies leñosas con un dap igual o superior a 5cm, además se obtuvo la ubicación espacial de cada individuo. Adicionalmente se realizaron 109 parcelas a lo largo de 4 de los cantones de la provincia de Loja que conservan en buen estado bosques secos. En el matorral seco se desarrollo un estudio observacional a lo largo de una gradiente estrés climático y dos niveles de manejo no disturbado y pastoreado. En cada nivel se colocaron 2 parcelas dentro de las cuales se registró la abundancia, diversidad y cobertura de todas las especies anuales. Además, se tomaron 9 muestras de suelo en cada parcela para evaluar el banco de semillas del suelo. Los resultados muestran que el bosque seco las relaciones positivas planta-planta juegan un papel fundamental en la estructura florística de las comunidades. Así se pudo observar que a escalas regionales la disponibilidad de agua tiene una relación inversa con la riqueza, encontrándose que áreas con menor disponibilidad de agua generan comunidades vegetales más diversas. Esta respuesta es coherente con la Hipótesis de la Gradiente de Estrés ya que el incremento de las relaciones de facilitación estaría incrementando la cantidad de especies que coexisten a nivel local. Por otro lado a escalas locales las especies de bosque seco tropical muestran un bajo grado de agrupamiento con sus congéneres, lo cual coincide con la propuesta de la “Dependencia Negativa de la Densidad” propuesta por Janzen – Connell. Esta respuesta contribuye al comportamiento acumulador de especies observado en el bosque seco, por lo observado en este trabajo a escalas locales las interacciones positivas planta-planta son predominantes. En el matorral seco montano los resultados muestran que la magnitud de los efectos de las especies ingenieras del ecosistema en la diversidad de especies y la productividad varían a lo largo de gradientes ambientales y a con la escala espacial de la comunidad. Así, en las zonas con mayor presión ambiental y una mayor presión de pastoreo, el efecto de las plantas ingenieras es mayor. Por otro lado, se pudo observar que la estabilidad de la diversidad en la escala de la comunidad parece ser la norma con efectos nulos de los factores de estrés en las propiedades de la comunidad. A nivel de microhábitat la importancia de la facilitación incrementa cuando los factores de estrés climático aumentan, y bajo las copas de Croton la diversidad y la cobertura es mayor que en el suelo desnudo, esta diferencia se incrementa con el estrés abiótico. Esta respuesta fue modulada por el pastoreo, en las zonas más altas con menor estrés climático, la facilitación se mantuvo debido a que la competencia se reduce por efecto del pastoreo, ya que las manchas se transforman refugios libres de herbivoría. Así, el estrés biótico por los consumidores altera significativamente la naturaleza y la fuerza de las interacciones entre especies en las comunidades, e incluso puede cambiar el signo de las interacciones entre especies. El banco de semillas muestra una importante interacción entre los factores de estrés, los efectos en el banco de semillas del suelo se explica mejor cuando los factores de estrés, tanto climáticos y el pastoreo son evaluados. En condiciones inalteradas la riqueza y abundancia del banco de semillas aumenta con la altitud y se reduce el estrés. Este patrón es modulado por el pastoreo, en el extremo inferior del gradiente altitudinal, el pastoreo produce un aumento en la riqueza y la densidad del banco de semillas y en el extremo superior del gradiente altitudinal el pastoreo reduce estos dos atributos.

Fungal microbiota from rain water and pathogenicity of Fusarium species isolated from atmospheric dust and rainfall dust

In order to determine the presence of Fusarium spp. in atmospheric dust and rainfall dust, samples were collected during September 2007, and July, August, and October 2008. The results reveal the prevalence of airborne Fusarium species coming from the atmosphere of the South East coast of Spain. Five different Fusarium species were isolated from the settling dust: Fusarium oxysporum, F. solani, F. equiseti, F. dimerum, and F. proliferatum. Moreover, rainwater samples were obtained during significant rainfall events in January and February 2009. Using the dilution-plate method, 12 fungal genera were identified from these rainwater samples. Specific analyses of the rainwater revealed the presence of three species of Fusarium: F. oxysporum, F. proliferatum and F. equiseti. A total of 57 isolates of Fusarium spp. obtained from both rainwater and atmospheric rainfall dust sampling were inoculated onto melon (Cucumis melo L.) cv. Piñonet and tomato (Lycopersicon esculentum Mill.) cv. San Pedro. These species were chosen because they are the main herbaceous crops in Almeria province. The results presented in this work indicate strongly that spores or propagules of Fusarium are able to cross the continental barrier carried by winds from the Sahara (Africa) to crop or coastal lands in Europe. Results show differences in the pathogenicity of the isolates tested. Both hosts showed root rot when inoculated with different species of Fusarium, although fresh weight measurements did not bring any information about the pathogenicity. The findings presented above are strong indications that long-distance transmission of Fusarium propagules may occur. Diseases caused by species of Fusarium are common in these areas. They were in the past, and are still today, a problem for greenhouses crops in Almería, and many species have been listed as pathogens on agricultural crops in this region. Saharan air masses dominate the Mediterranean regions. The evidence of long distance dispersal of Fusarium spp. by atmospheric dust and rainwater together with their proved pathogenicity must be taken into account in epidemiological studies.

Fine spatial pattern of an epiphytic lichen species is affected by habitat conditions in two forest types in the Iberian Mediterranean region

Persistence and abundance of species is determined by habitat availability and the ability to disperse and colonize habitats at contrasting spatial scales. Favourable habitat fragments are also heterogeneous in quality, providing differing opportunities for establishment and affecting the population dynamics of a species. Based on these principles, we suggest that the presence and abundance of epiphytes may reflect their dispersal ability, which is primarily determined by the spatial structure of host trees, but also by host quality. To our knowledge there has been no explicit test of the importance of host tree spatial pattern for epiphytes in Mediterranean forests. We hypothesized that performance and host occupancy in a favourable habitat depend on the spatial pattern of host trees, because this pattern affects the dispersal ability of each epiphyte and it also determines the availability of suitable sites for establishment. We tested this hypothesis using new point pattern analysis tools and generalized linear mixed models to investigate the spatial distribution and performance of the epiphytic lichen Lobaria pulmonaria, which inhabits two types of host trees (beeches and Iberian oaks). We tested the effects on L. pulmonaria distribution of tree size, spatial configuration, and host tree identity. We built a model including tree size, stand structure, and several neighbourhood predictors to understand the effect of host tree on L. pulmonaria. We also investigated the relative importance of spatial patterning on the presence and abundance of the species, independently of the host tree configuration. L. pulmonaria distribution was highly dependent on habitat quality for successful establishment, i.e., tree species identity, tree diameter, and several forest stand structure surrogates. For beech trees, tree diameter was the main factor influencing presence and cover of the lichen, although larger lichen-colonized trees were located close to focal trees, i.e., young trees. However, oak diameter was not an important factor, suggesting that bark roughness at all diameters favoured lichen establishment. Our results indicate that L. pulmonaria dispersal is not spatially restricted, but it is dependent on habitat quality. Furthermore, new spatial analysis tools suggested that L. pulmonaria cover exhibits a distinct pattern, although the spatial pattern of tree position and size was random.

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.

Genome-Wide Analysis of the Response of Dickeya dadantii 3937 to Plant Antimicrobial Peptides

Antimicrobial peptides constitute an important factor in the defense of plants against pathogens, and bacterial resistance to these peptides have previously been shown to be an important virulence factor in Dickeya dadantii, the causal agent of soft-rot disease of vegetables. In order to understand the bacterial response to antimicrobial pep- tides, a transcriptional microarray analysis was performed upon treatment with sub-lethal concentration of thionins, a widespread plant peptide. In all, 36 genes were found to be overexpressed, and were classified according to their deduced function as i) transcriptional regulators, ii) transport, and iii) modification of the bacterial membrane. One gene encoding a uricase was found to be repressed. The majority of these genes are known to be under the control of the PhoP/PhoQ system. Five genes representing the different functions induced were selected for further analysis. The results obtained indicate that the presence of antimicrobial peptides induces a complex response which includes peptide-specific elements and general stress-response elements contributing differentially to the virulence in different hosts.