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.

Dinámica vegetal desde el tardiglaciar en la cuenca del Duero: interacciones con clima, fuego e impacto humano

La cuenca del Duero constituye un vasto territorio ibérico cuyo paisaje se encuentra actualmente muy alterado por la mano del hombre y es prácticamente imposible localizar alguna manifestación de su cubierta vegetal natural. Aunque la historia de la vegetación en los sectores central y oriental es relativamente bien conocida, en su mitad occidental los registros paleoecológicos estudiados hasta la fecha son prácticamente inexistentes. Esto hace que se desconozca la respuesta de la vegetación a las diferentes oscilaciones climáticas que se han producido desde el Último Máximo Glaciar, cuál fue el impacto de las diferentes culturas sobre el medio, cuándo se produjo una alteración profunda de la vegetación natural y cuál ha sido la historia de los incendios. Este último aspecto, el papel e importancia de los incendios, reviste un especial interés en la península Ibérica dada su situación geográfica y climática dentro de la cuenca Mediterránea, donde el fuego es un factor ecológico de primer nivel. Las distintas técnicas paleoecológicas son las más adecuadas para abordar todas estas preguntas. De este modo, los avatares de la vegetación a través del tiempo se han reconstruido mediante el análisis polínico y de macrofósiles, el impacto humano se ha trazado utilizando indicadores polínicos ligados a actividades antrópicas y esporas de hongos coprófilos, estudiándose los incendios a partir del registro de partículas microscópicas de carbón. La alta resolución temporal y taxonómica alcanzada en estos análisis, así como la amplia superficie abarcada con los yacimientos estudiados, hacen que la información obtenida sea altamente detallada y complete el conocimiento que se tiene sobre la cuenca del Duero. El Tardiglaciar se registra por primera vez en la Meseta Norte Ibérica en la secuencia de Ayoó de Vidriales, donde una vegetación esteparia prácticamente desarbolada domina durante los periodos fríos. Durante el interestadial Bølling/Allerød la expansión forestal (pinos, abedules) fue muy leve y tardía y fue interrumpida bruscamente por el Dryas Reciente. Al final del Dryas Reciente o al inicio del Holoceno se produjo una acusada y rápida expansión de los bosques. Esta dinámica sugiere que no hubo refugios glaciares importantes en esta zona de la Meseta durante el “Mystery Interval”, aparentemente el periodo más frío y seco. Los incendios fueron muy escasos, aumentando de forma muy brusca al inicio del Holoceno por el aumento de biomasa y las condiciones relativamente cálidas y secas. A partir de los registros de Ayoó y El Maíllo se consolida la importancia del gradiente oceanicidad-continentalidad en el Sistema Central y la Meseta Norte, que se manifiesta principalmente en la dominancia más prolongada de los pinares hacia el interior. Además, otra de las principales contribuciones de la presente tesis es proporcionar evidencia sobre la sucesión de distintos tipos de bosques en el noroeste de la Meseta, precisando el marco temporal en el que suceden. Así, se ha constatado que hubo un máximo desarrollo del bosque caducifolio durante el Holoceno medio en Ayoó de Vidriales y una baja incidencia del fuego que sugieren que el clima fue más húmedo que en el Holoceno inicial. El estudio de macrofósiles leñosos ha permitido detectar procesos que con el análisis polínico habrían pasado desapercibidos, como la persistencia hasta el Holoceno final de Pinus sylvestris en la sierra del Teleno y la sustitución de P. sylvestris por P. pinaster en la sierra de Francia durante el Holoceno inicial. También el estudio de los carbones procedentes de los arenales de Tierra de Pinares han proporcionado la prueba definitiva de la naturalidad de los pinares de P. pinaster. El impacto humano se detecta temprano en las secuencias del oeste de la cuenca del Duero, durante el Neolítico, aunque ha sido mucho más acusado desde la Edad del Hierro (ca 2700-2500 años cal BP). Para la detección del impacto humano temprano ha sido clave el análisis de esporas de hongos coprófilos, cuyo análisis fue incorporado en la secuencia de Ayoó de Vidriales. Una de sus principales consecuencias fue el establecimiento de comunidades de matorral (brezales, piornales) sobre amplias extensiones del occidente de la cuenca del Duero, vinculado al recrudecimiento de los regímenes de incendios. A pesar de que los incendios han sido ecológicamente importantes desde el inicio del Holoceno, los cambios introducidos por el hombre en sus regímenes sobrepasaron la resiliencia de los bosques originales, lo cual condujo a su sustitución sostenida por matorrales. ABSTRACT The Duero Basin constitutes a vast Iberian territory which is currently strongly disturbed due to human activities, so it is very difficult to find any remnant of the natural vegetation. Vegetation history for the eastern and western sectors of the Basin is relatively well-known but, in contrast, there is an almost complete lack of palaeoecological record in the western area. Consequently, there exists a profound ignorance about vegetation responses to the climatic oscillations occurred since the Last Glacial Maximum, the environmental impact of the different cultures, when a severe disturbance of the natural vegetation took place and fire history. The last question, the role and importance of fire, has a special interest in the Iberian Peninsula due to its geographic and climatic framework, the Mediterranean Basin, where fire is a major ecological factor. The diverse palaeoecological techmiques are the most suitable tools to address all these questions. Thus, vegetation shifts through time have been reconstructed using pollen and macrofossil analyses, human impact has been tracked by means of anthropogenic pollen indicators and dung fungal spores, while fire history has been studied from the quantification of microscopic charcoal particles. The high taxonomic and time resolution attained along with the extensive surface covered by the studied sites provide detailed information very useful to complete the knowledge on landscape dynamics in the Duero Basin. The Lateglacial is recorded for the first time in the Northern Iberian Plateau in the sequence from Ayoó de Vidriales, showing that almost treeless steppic vegetation dominated during the cold periods. Tree expansion (pines, birches) was late and slight during the Bølling/Allerød interstadial and was sharply interrupted by the Younger Dryas (YD) climatic reversal. By the end of the YD or the onset of the Holocene, a rapid forest expansion occurred. This forest dynamics suggests an absence of important glacial refugia for trees in this area of the Plateau during the Mystery Interval, apparently the coldest and driest period. Fires were fairly rare, increasing abruptly at the beginning of the Holocene due to the relatively warm and dry climate and the accumulation of biomass. The records from Ayoó and El Maíllo reinforce the role of the oceanicity-continentality gradient in the vegetation history of the Iberian Central Range and the Iberian Northern Plateau, reflected mainly in the longer dominance of pine forests towards inland areas. Further, another important contribution of this PhD Thesis is providing evidence on the succession of different forest types in the northestern fringe of the Plateau, specifying the chronological framework. A maximum of deciduous forest development and low fire activity have been detected in Ayoó de Vidriales during the mid-Holocene, suggesting that climate was wetter than in the early Holocene. The study of woody macrofossils has allowed detecting processes which would have remained unnoticed using pollen analysis alone, such as the persistence of Pinus sylvestris until the late Holocene in the Teleno Mountains and the early Holocene replacement of P. sylvestris with P. pinaster in the sierra de Francia range. The study of macroscopic charcoal fragments from palaeosols of the Tierra de Pinares has also provided the definitive proof of naturalness for the P. pinaster stands gorwing over this area at present. Early human impact, during the Neolithic, has been detected in the sequences from the western sector of the Duero Basin, although human disturbance has been more severe from the Iron Age onwards (ca 2700-2500 cal yr BP). The analysis of coprophilous fungi incorporated in the sequence of Ayoó de Vidriales has played a key role in recognizing that early human impact. One of the main consequences of human disturbance was the establishment of shrubland communities (heaths, brooms) over huge areas of the western Duero Basin, linked to severe and/or frequent fires. Despite fires has been ecologically important since the onset of the Holocene, human-induced changes in fire regimes have exceeded the resilience of original forests leading to a sustained replacement with shrublands.

Effect of solute and matric potential on growth rate of fungal species isolated from cheese

The effect of water potential ( J w ) on the growth of 15 fungal species isolated from cheeses was analysed. The species, identified mainly by analysis of DNA sequences, belonged to genera Penicillium , Geotrichum , Mucor , Aspergillus , Microascus and Talaromyces . Particularly, the effect of matric potential ( J m ), and ionic (NaCl) and non-ionic (glycerol) solute potentials ( J s ) on growth rate was studied. The response of strains was highly dependent on the type of J w . For J s , clear profiles for optimal, permissive and marginal conditions for growth were obtained, and differences in growth rate were achieved comparing NaCl and glycerol for most of the species. Conversely, a sustained growth was obtained for J m in all the strains, with the exception of Aspergillus pseudoglaucus , whose growth increased proportionally to the level of water stress. Our results might help to understand the impact of environmental factors on the ecophysiology and dynamics of fungal populations associated to cheeses.

Fungal biodeterioration of colour cinematographic films of the cultural heritage of Cuba

Until recently, cinematographic film was largely cellulose-triacetate-based. However, this material is highly susceptible to biodeterioration, thus placing historic film collections, an important part of the cultural heritage of many countries, at risk. In the present study, samples taken from several biodeteriorated color cinematographic films belonging to the collection of the Cuban Institute for Cinematographic Industry and Arts (ICAIC) were investigated. Infrared spectroscopy showed that all films were of the same composition, i.e., a gelatin emulsion coating one side of a cellulose-triacetate-based film support. The films were analyzed by environmental scanning electron microscopy and scanning electron microscopy to determine the degree of biodeterioration and the type of colonizing microorganisms. Significant fungal colonization was found on both sides of the films in all samples, with a higher concentration of fungi on the gelatin emulsion side. Epifluorescence microscopy of fluorochrome-dyed films demonstrated that some of the fungi were still active, indicating that the films under study, and probably others at the ICAIC, are at risk of further deterioration. Fungi were identified by molecular biology techniques. The fungi mainly responsible for the observed biodeterioration were those belonging to the genera Aspergillus and Cladosporium, although other genera, such as Microascus and Penicillium, were identified as well. In accordance with the findings described herein, the existing guidelines for the prevention and control of film biodeterioration are discussed.

Effect of solute and matric potential on growth rate of fungal species isolated from cheese

The effect of water potential ( J w ) on the growth of 15 fungal species isolated from cheeses was analysed. The species, identi fi ed mainly by analysis of DNA sequences, belonged to genera Penicillium, Geotrichum, Mucor , Aspergillus , Microascus and Talaromyces . Particularly, the effect of matric potential ( J m ), and ionic (NaCl) and non-ionic (glycerol) solute potentials ( J s ) on growth rate was studied. The response of strains was highly dependent on the type of J w . For J s, clear profiles for optimal, permissive and marginal conditions for growth were obtained, and differences in growth rate were achieved comparing NaCl and glycerol for most of the species. Conversely, a sustained growth was obtained for J m in all the strains, with the exception of Aspergillus pseudoglaucus, whose growth increased proportionally to the level of water stress. Our results might help to understand the impact of environmental factors on the ecophysiology and dynamics of fungal populations associated to cheeses.

Analysis of the role of the Spitzenkörper in fungal morphogenesis by computer simulation of apical branching in Aspergillus niger

High-resolution video microscopy, image analysis, and computer simulation were used to study the role of the Spitzenkörper (Spk) in apical branching of ramosa-1, a temperature-sensitive mutant of Aspergillus niger. A shift to the restrictive temperature led to a cytoplasmic contraction that destabilized the Spk, causing its disappearance. After a short transition period, new Spk appeared where the two incipient apical branches emerged. Changes in cell shape, growth rate, and Spk position were recorded and transferred to the fungus simulator program to test the hypothesis that the Spk functions as a vesicle supply center (VSC). The simulation faithfully duplicated the elongation of the main hypha and the two apical branches. Elongating hyphae exhibited the growth pattern described by the hyphoid equation. During the transition phase, when no Spk was visible, the growth pattern was nonhyphoid, with consecutive periods of isometric and asymmetric expansion; the apex became enlarged and blunt before the apical branches emerged. Video microscopy images suggested that the branch Spk were formed anew by gradual condensation of vesicle clouds. Simulation exercises where the VSC was split into two new VSCs failed to produce realistic shapes, thus supporting the notion that the branch Spk did not originate by division of the original Spk. The best computer simulation of apical branching morphogenesis included simulations of the ontogeny of branch Spk via condensation of vesicle clouds. This study supports the hypothesis that the Spk plays a major role in hyphal morphogenesis by operating as a VSC—i.e., by regulating the traffic of wall-building vesicles in the manner predicted by the hyphoid model.

The roles of zinc and copper sensing in fungal pathogenesis

Open Access funded by Wellcome Trust Acknowledgements ERB is funded by the BBSRC (BB/M014525/1). DW is supported by a Sir Henry Dale Fellowship jointly funded by the Wellcome Trust and the Royal Society (Grant Number 102549/Z/13/Z). We additionally acknowledge the MRC and University of Aberdeen for funding (MR/N006364/1) and the Wellcome Trust Strategic Award for Medical Mycology and Fungal Immunology (097377/Z/11/Z). Finally, we acknowledge FungiDB and the Candida Genome Database [ 56 and 57].

Host-Imposed Copper Poisoning Impacts Fungal Micronutrient Acquisition during Systemic Candida albicans Infections

This work was supported by the European Research Council (http://erc.europa.eu/: STRIFE Advanced Grant ERC-2009-AdG-249793). A.J.P.B. was also supported by the UK Biotechnology and Biological Research Council (www.bbsrc.ac.uk: Research Grants BB/F00513X/1, BB/K017365/1), the UK Medical Research Council (www.mrc.ac.uk: Programme Grant MR/M026663/1; Centre Grant MR/ N006364/1), and the Wellcome Trust (www.wellcome.ac.uk: Strategic Award 097377)

A Fungal Kinesin Required for Organelle Motility, Hyphal Growth, and Morphogenesis

A gene (NhKIN1) encoding a kinesin was cloned from Nectria haematococca genomic DNA by polymerase chain reaction amplification, using primers corresponding to conserved regions of known kinesin-encoding genes. Sequence analysis showed that NhKIN1 belongs to the subfamily of conventional kinesins and is distinct from any of the currently designated kinesin-related protein subfamilies. Deletion of NhKIN1 by transformation-mediated homologous recombination caused several dramatic phenotypes: a 50% reduction in colony growth rate, helical or wavy hyphae with reduced diameter, and subcellular abnormalities including withdrawal of mitochondria from the growing hyphal apex and reduction in the size of the Spitzenkörper, an apical aggregate of secretory vesicles. The effects on mitochondria and Spitzenkörper were not due to altered microtubule distribution, as microtubules were abundant throughout the length of hyphal tip cells of the mutant. The rate of spindle elongation during anaphase B of mitosis was reduced 11%, but the rate was not significantly different from that of wild type. This lack of a substantial mitotic phenotype is consistent with the primary role of the conventional kinesins in organelle motility rather than mitosis. Our results provide further evidence that the microtubule-based motility mechanism has a direct role in apical transport of secretory vesicles and the first evidence for its role in apical transport of mitochondria in a filamentous fungus. They also include a unique demonstration that a microtubule-based motor protein is essential for normal positioning of the Spitzenkörper, thus providing a new insight into the cellular basis for the aberrant hyphal morphology.

Muramic lactam in peptidoglycan of Bacillus subtilis spores is required for spore outgrowth but not for spore dehydration or heat resistance

Bacterial endospores derive much of their longevity and resistance properties from the relative dehydration of their protoplasts. The spore cortex, a peptidoglycan structure surrounding the protoplasm, maintains, and is postulated to have a role in attaining, protoplast dehydration. A structural modification unique to the spore cortex is the removal of all or part of the peptide side chains from the majority of the muramic acid residues and the conversion of 50% of the muramic acid to muramic lactam. A mutation in the cwlD gene of Bacillus subtilis, predicted to encode a muramoyl-l-alanine amidase, results in the production of spores containing no muramic lactam. These spores have normally dehydrated protoplasts but are unable to complete the germination/outgrowth process to produce viable cells. Addition of germinants resulted in the triggering of germination with loss of spore refractility and the release of dipicolinic acid but no degradation of cortex peptidoglycan. Germination in the presence of lysozyme allowed the cwlD spores to produce viable cells and showed that they have normal heat resistance properties. These results (i) suggest that a mechanical activity of the cortex peptidoglycan is not required for the generation of protoplast dehydration but rather that it simply serves as a static structure to maintain dehydration, (ii) demonstrate that degradation of cortex peptidoglycan is not required for spore solute release or partial spore core rehydration during germination, (iii) indicate that muramic lactam is a major specificity determinant of germination lytic enzymes, and (iv) suggest the mechanism by which the spore cortex is degraded during germination while the germ cell wall is left intact.