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Infectious diseases are among the top causes of mortality and a leading cause of disability worldwide. Drug-resistant bacterial infections are estimated to directly cause 1.27 million deaths and to contribute to approximately 4.95 million deaths every year, with the greatest burden in resource- limited settings. Against the backdrop of this major global health threat, invasive fungal diseases (IFDs) are rising overall and particularly among immunocompromised populations. The diagnosis and treatment of IFDs are challenged by limited access to quality diagnostics and treatment as well as emergence of antifungal resistance in many settings. Despite the growing concern, fungal infections receive very little attention and resources, leading to a paucity of quality data on fungal disease distribution and antifungal resistance patterns. Consequently, it is impossible to estimate their exact burden. In 2017, WHO developed its first bacterial priority pathogens list (WHO BPPL) in the context of increasing antibacterial resistance to help galvanize global action, including the research and development (R&D) of new treatments. Inspired by the BPPL, WHO has now developed the first fungal priority pathogens list (WHO FPPL). The WHO FPPL is the first global effort to systematically prioritize fungal pathogens, considering their unmet R&D needs and perceived public health importance. The WHO FPPL aims to focus and drive further research and policy interventions to strengthen the global response to fungal infections and antifungal resistance. The development of the list followed a multicriteria decision analysis (MCDA) approach. The prioritization process focused on fungal pathogens that can cause invasive acute and subacute systemic fungal infections for which drug resistance or other treatment and management challenges exist. The pathogens included were ranked, then categorized into three priority groups (critical, high, and medium). The critical group includes Cryptococcus neoformans, Candida auris, Aspergillus fumigatus and Candida albicans. The high group includes Nakaseomyces glabrata (Candida glabrata), Histoplasma spp., eumycetoma causative agents, Mucorales, Fusarium spp., Candida tropicalis and Candida parapsilosis. Finally, pathogens in the medium group are Scedosporium spp., Lomentospora prolificans, Coccidioides spp., Pichia kudriavzeveii (Candida krusei), Cryptococcus gattii, Talaromyces marneffei, Pneumocystis jirovecii and Paracoccidioides spp. This document proposes actions and strategies for policymakers, public health professionals and other stakeholders, targeted at improving the overall response to these priority fungal pathogens, including preventing the development of antifungal drug resistance. Three primary areas for action are proposed, focusing on: (1) strengthening laboratory capacity and surveillance; (2) sustainable investments in research, development, and innovation; and (3) public health interventions. Countries are encouraged to improve their mycology diagnostic capacity to manage fungal infections and to perform surveillance. In most contexts, this might require a stepwise approach. There is a need for sustainable investments in research, development, and innovation. More investments are needed in basic mycology research, R&D of antifungal medicines and diagnostics. Innovative approaches are needed to optimize and standardize the use of current diagnostic modalities globally. In addition, public health interventions are needed to highlight the importance of fungal infections, including through incorporating fungal diseases and priority pathogens in medical (clinical) and public health training programmes and curricula at all levels of training. Similarly, collaboration across sectors is required to address the impact of antifungal use on resistance across the One Health spectrum. Finally, regional variations and national contexts need to be taken into consideration while implementing the WHO FPPL to inform priority actions. Sí

Fil: Depetris, Mara Belén. Universidad Nacional del Nordeste. Facultad de Ciencias Agrarias; Argentina. Fil: Felitti, Silvina Andrea. Universidad Nacional del Nordeste. Facultad de Ciencias Agrarias; Argentina. Fil: Acuña, Carlos Alberto. Universidad Nacional del Nordeste. Facultad de Ciencias Agrarias; Argentina. En los ecosistemas naturales, las plantas parecen estar en simbiosis con diversos hongos endofitos que pueden tener impactos en las comunidades vegetales. Paspalum es uno de los géneros de gramíneas más importantes de los pastizales de América del Sur. El objetivo de este trabajo fue determinar la presencia de hongos endofitos en dos especies de Paspalum y evaluar sus efectos sobre el crecimiento de las plantas y sobre los hongos patógenos causantes de enfermedades. Se recolectaron ochenta y cuatro muestras de plantas de Paspalum notatum en un pastizal en el noreste de Argentina, y sesenta de Paspalum dilatatum en el centro de Argentina. Se realizó un proceso de triple esterilización sumergiendo pequeños trozos de hojas y tallos para el aislamiento de hongos endofitos de las plantas recolectadas. Para la identificación se analizaron los caracteres morfológicos y las secuencias fúngicas correspondientes a la región ITS (Internal Transcribed Spacer). Se identificaron un total de diez géneros: Acremonium, Alternaria, Aspergillus, Bipolaris, Cercospora, Cladosporium, Curvularia, Fusarium, Penicillium y Trichoderma. Se realizó un ensayo en invernadero para determinar los efectos de los hongos endofitos sobre el crecimiento vegetal. El tratamiento con agua caliente eliminó los hongos preexistentes de las semillas. Las libres de endofitos (E-) se esterilizaron en la superficie y se germinaron asépticamente en placas de Petri. La mitad de las plántulas E- se inocularon para obtener plantas E+. Los resultados mostraron que Trichoderma amazonicum aumentó significativamente el contenido relativo de clorofila en P. notatum y Acremonium strictum el número de macollos y la biomasa aérea y radical en P. dilatatum. A su vez, se realizaron pruebas de campo y laboratorio para determinar los efectos de los hongos endofitos sobre el patógeno Claviceps paspali. Se observó un efecto significativo de los dos endofitos evaluados (T. amazonicum y A. strictum), aumentando el número de semillas sanas y el peso en ambos forrajes estudiados. En conclusión, se logró describir la diversidad de hongos endofitos presentes y se demostró que algunos aspectos del crecimiento y del control de patógenos fúngicos en Paspalum parecen verse afectados positivamente por las especies T. amazonicum y A. strictum In natural ecosystems, plants appear to be in symbiosis with diverse fungal endophytes which can have impacts on plant communities. Paspalum is one of the most important grass genera in South American grasslands. The aim of this work was to determine the presence of fungal endophytes in two Paspalum species and evaluate their effects on plant growth and on disease-causing fungal pathogens. Eighty-four plant samples were collected from Paspalum notatum in a rangeland in northeastern Argentina, and sixty for Paspalum dilatatum in central Argentina. A triple-sterilization process was carried out by submerging small pieces of leaves and stems for the isolation of fungi from the collected plants. In order to identify endophytes, morphological characters and fungal sequences corresponding to the ITS (Internal Transcribed Spacer) region were analysed. A total of ten genera were identified: Acremonium, Alternaria, Aspergillus, Bipolaris, Cercospora, Cladosporium, Curvularia, Fusarium, Penicillium and Trichoderma. A greenhouse trial was conducted to determine the effects of fungal endophytes on growth. A hot water treatment removed preexisting endophytic fungi from seeds. Endophyte free seeds (E-) were surfacesterilized and aseptically germinated in Petri plates. The half of E- seedlings were inoculated to obtain E+ plants. The results showed that Trichoderma amazonicum significantly increased relative chlorophyll content in P. notatum and Acremonium strictum increased tiller number, and above and below ground biomass in P. dilatatum. Moreover, field and lab trials were performed in order to determine fungal endophytes effects on the pathogen Claviceps paspali. A significant effect of the two endophytes evaluated has observed (T. amazonicum and A. strictum), increasing the number of healthy seeds and weight in both forages studied. In conclusion, the diversity of fungal endophytes present was described and some aspects of growth and fungal pathogens control in Paspalum seem to be positively affected by T. amazonicum y A. strictum.

Pulsed light, as a postharvest technology, is an alternative to traditional fungicides, and can be used on a wide variety of fruit and vegetables for sanitization or pathogen control. In addition to these applications, other effects also are detected in vegetal cells, including changes in metabolism and secondary metabolite production, which directly affect disease control response mechanisms. This study aimed to evaluate pulsed ultraviolet light in controlling postharvest rot, caused by Fusarium pallidoroseum in 'Spanish' melon, in natura, and its implications in disease control as a function of metabolomic variation to fungicidal or fungistatic effects. The dose of pulsed light (PL) that inhibited F. pallidoroseum growth in melons (Cucumis melo var. Spanish) was 9 KJ m-2. Ultra-performance liquid chromatography (UPLC) coupled to a quadrupole-time-of-flight (QTOF) mass analyzer identified 12 compounds based on tandem mass spectrometry (MS/MS) fragmentation patterns. Chemometric analysis by Principal Components Analysis (PCA) and Orthogonal Partial Least Squared Discriminant Analysis (OPLS-DA) and corresponding S-Plot were used to evaluate the changes in fruit metabolism. PL technology provided protection against postharvest disease in melons, directly inhibiting the growth of F. pallidoroseum through the upregulation of specific fruit biomarkers such as pipecolic acid (11), saponarin (7), and orientin (3), which acted as major markers for the defense system against pathogens. PL can thus be proposed as a postharvest technology to prevent chemical fungicides and may be applied to reduce the decay of melon quality during its export and storage.

Submitted by Lucas Antonio Benso (lucas_benso@hotmail.com) on 2019-08-12T13:04:39Z No. of bitstreams: 1 Dissertacao_Lucas_Benso_2019_Pronta.pdf: 4426413 bytes, checksum: 3be8f9a44aedafdac506dadb054f0b78 (MD5) Approved for entry into archive by Ana Lucia de Grava Kempinas (algkempinas@fca.unesp.br) on 2019-08-12T14:29:20Z (GMT) No. of bitstreams: 1 benso_la_me_botfca_par.pdf: 866120 bytes, checksum: 9d7a0726a63fee4866a71f1193189ab4 (MD5) Made available in DSpace on 2019-08-12T14:29:20Z (GMT). No. of bitstreams: 1 benso_la_me_botfca_par.pdf: 866120 bytes, checksum: 9d7a0726a63fee4866a71f1193189ab4 (MD5) Previous issue date: 2019-06-28 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) A recente introdução de diversas espécies florestais exóticas no Brasil, na forma plantios comerciais, é responsável pela redução do dano ocasionado pela exploração desenfreada em áreas naturais. As espécies de mogno africano (Khaya spp.) estão entre aquelas introduzidas há pouco tempo, visando principalmente suprir a demanda de mercado pelo mogno brasileiro (Swietenia macrophylla). A falta de estudos sobre essas árvores exóticas é um dos fatores que dificultam o seu manejo, principalmente do ponto de vista fitossanitário. Esse trabalho teve como objetivo o estudo de pragas e doenças em duas espécies de mogno africano, Khaya senegalensis e Khaya grandifoliola. No primeiro capítulo, foi realizado o estudo da dispersão espacial da murcha de Ceratocystis em K. senegalensis. Para isso, três parcelas foram montadas em um talhão puro dessa espécie florestal. Foram feitas avaliações anuais ao longo de três anos, com a contagem do número de plantas doentes e a elaboração de mapas com sua distribuição. Esses mapas serviram de base para a aplicação da Análise da Dinâmica e Estrutura de Focos (ADEF) e do Indice de Dispersão (ID). Para o primeiro, a murcha apresentou padrão tipo “agregado” em todas as parcelas e em todos os anos avaliados, sem preferência entre linha ou coluna para o crescimento dos focos. Já para o ID, a dispersão variou entre “agregado” e “ao acaso”, podendo se notar que quanto maiores eram os quadrats usados, mais predominantes eram os resultados com distribuição “agregado”, enquanto que com a redução dos mesmos, predominaram os “ao acaso”. No segundo capítulo foi feito o levantamento de doenças e a identificação dos seus agentes causais em um viveiro de mudas de K. senegalensis e K. grandifoliola. No total, três novas doenças foram encontradas, todas associadas a manchas foliares. Em K. grandifoliola, duas doenças tiveram seus agentes causais isolados, uma mais severa causada por Boeremia exigua, e outra com incidência principalmente em locais sombreados, causada por Epicoccum sorghinum. Em K. senegalensis, o fungo B. exigua foi o único associado com manchas foliares. A inoculação desses patógenos em folíolos destacados das duas espécies de Khaya demonstrou a possibilidade de transmissão de E. sorghinum para K. senegalensis. Já no terceiro capítulo, o objetivo foi realizar o levantamento de coleobrocas em talhões de diferentes idades de K. senegalensis e relacionar as espécies encontradas com altura de ataque, idade das árvores, sanidade do tecido atacado e capacidade da planta de se defender. Para isso foram feitas coletas bimestrais durante um ano, sendo possível notar certas variações nas ocorrências de determinadas espécies de coleobrocas de acordo com as características do hospedeiro levantadas. Dentre as espécies encontradas, Megaplatypus dentatus foi coletada apenas do interior de tecidos sadios, enquanto Euplatypus parallelus e Theoborus villosulus foram retirados principalmente de tecidos sadios e estressados. As demais espécies coletadas foram encontradas em tecidos mortos e/ou estressados em sua maior parte, devido a doenças associadas ao sistema radicular. The recent introduction of several exotic forest species in Brazil, in the form of commercial plantations, is responsible for reducing the damage caused by uncontrolled exploitation in natural areas. The species of African mahogany (Khaya spp.) are among those recently introduced mainly to supply the market demand for Brazilian mahogany (Swietenia macrophylla). The lack of studies on these exotic trees is one of the factors that make their management difficult, especially from a phytosanitary point of view. This work had as objective the study of pests and diseases in two species of African mahogany, Khaya senegalensis and Khaya grandifoliola. In the first chapter, the study of the spatial dispersion of Ceratocystis wilt in K. senegalensis was carried out. For this, three parcels were mounted on a pure plot of this species. Annual evaluations were carried out over three years, counting the number of diseased plants and drawing maps with their distribution. These maps served as a basis for the application of the Dynamics and Structure Analysis of Foci technique (DSAF) and Dispersion Index (DI). For the first technique, the wilt showed an "aggregate" pattern in all plots and in all evaluated years, with no preference between row or column for growth of foci. For DI, the dispersion ranged from "aggregate" to "random" and it may be noted that the larger the quadrats used, the more "aggregate" results were predominant, while with the reduction the “random” pattern predominated. In the second chapter was made a disease surveyed and the identification of its causal agents in a seedling nursery of K. senegalensis and K. grandifoliola. In total, three new diseases were found, all associated with leaf spots. In K. grandifoliola, two diseases had their causal agents isolated, one more severe caused by Boeremia exigua, and another occurring mainly in shaded places, caused by Epicoccum sorghinum. In K. senegalensis, B. exigua fungus was the only one associated with leaf spot. The inoculation of these pathogens on detached leaves of the two species of Khaya demonstrated the possibility of transmission of E. sorghinum to K. senegalensis. In the third chapter the objective was to carry out a survey of coleobrocans in different ages of K. senegalensis, and to relate the species found with attack height, age of the trees, tissue sanity and plant capacity to defend themselves. For this, bimonthly collections were made during one year, and it is possible to observe certain variations in the occurrences of certain coleobroca species according to the characteristics of the host raised. Among the species found, Megaplatypus dentatus was collected only from the interior of healthy tissues, while Euplatypus parallelus and Theoborus villosulus were collected mainly from healthy and stressed tissues. The other species collected were found in dead and/or stressed tissues, mostly due to diseases associated with the root system.

Resumo: O objetivo deste trabalho foi avaliar a eficiência da aplicação foliar de fungicidas na redução da incidência de patógenos fúngicos, de grãos ardidos e de fumonisinas totais em grãos de milho (Zea mays). Foram conduzidos três ensaios: um no município de Luís Eduardo Magalhães, na Bahia, e dois em Sete Lagoas, em Minas Gerais, nas safras 2010/2011 e 2011/2012, com cultivo de diferentes híbridos comerciais. Os seguintes fungicidas foram aplicados zero, uma e duas vezes: picoxistrobina + ciproconazol, piraclostrobina + epoxiconazole, trifloxistrobina + tebuconazole, azoxistrobina + ciproconazol e tiofanato metílico. Em todos os ensaios, foram realizados a quantificação da incidência de grãos ardidos, o teste de patologia de sementes e a quantificação dos teores de fumonisinas totais. Não foi observada diferença significativa para os fatores fungicida e número de aplicações, exceto para rendimento de grãos, em todos os ensaios. Esses resultados são indicativos da ineficiência da aplicação de fungicidas na redução da incidência de patógenos fúngicos, de grãos ardidos e dos teores de fumonisinas totais em grãos de milho. Abstract: The objective of this work was to evaluate fungicide leaf application on the reduction of fungal pathogen incidence, kernel rot, and total fumonisins in corn (Zea mays) kernels. Three experiments were performed: one in the municipality of Luís Eduardo Magalhães, in the state of Bahia, and two in the municipality of Sete Lagoas, in the state of Minas Gerais, Brazil, during the 2010/2011 and 2011/2012 crop seasons, with planting of different commercial hybrids. The following fungicides were applied zero, one, and two times: picoxystrobin + cyproconazole, pyraclostrobin + epoxiconazole, trifloxystrobin + tebuconazole, azoxystrobin + cyproconazole, and thiophanate-methyl. For all trials, the quantification of kernel rot incidence, the grain pathology test, and the quantification of total fumonisins were performed. No significant difference was observed for the factors fungicide and number of applications, except for grain yield, in all trials. These results show the ineffectiveness of fungicide applications in reducing fungi incidence, kernel rot, and levels of total fumonisins in corn kernels.

Los patógenos fúngicos representan una grave amenaza para la salud humana y la seguridad alimentaria. Fusarium oxysporum es un hongo filamentoso que causa marchitez vascular en más de cien cultivos diferentes y es conocido por su amplia distribución ecológica a nivel mundial. A pesar de carecer de reproducción sexual conocida, posee una extraordinaria plasticidad genética. Reconocido como primer modelo fúngico de patógeno multihospedador, tiene la capacidad de infectar organismos tanto del reino vegetal como animal, en este último caso como patógeno oportunista. Esta gran variabilidad le permite adaptarse y colonizar nichos muy diferentes convirtiéndolo en un organismo ideal para estudiar mecanismos genéticos adaptativos. En este estudio se llevaron a cabo experimentos de evolución experimental de F. oxysporum en tres condiciones distintas: infección de plantas, placas de medio mínimo y placas de medio completo. Con ello se pretendía estudiar, como la presión selectiva ejercida por estas condiciones ambientales impulsara cambios genéticos que pudieran manifestarse a nivel fenotípico. A partir de la cepa original clónica 4287 de F. oxysporum f. sp lycopersici se obtuvieron 5 líneas independientes evolucionadas en cadas condición mediante diez pasajes seriados. La re-secuenciación del genoma de las poblaciones evolucionadas tras el último pasaje reveló la presencia de grandes duplicaciones segmentarias y deleciones en regiones accesorias del genoma, incluida la pérdida parcial de cromosomas. Además, se detectaron numerosas inserciones de transposones, así como algunos cambios de un solo nucleótido (SNPs) y pequeñas inserciones o deleciones (Indels). El seguimiento de estos eventos a lo largo del experimento, a través de diversas técnicas moleculares, permitió establecer la dinámica evolutiva dentro de las poblaciones, relacionada con la competitividad relativa de las mutaciones múltiples propagadas simultáneamente. Algunas de las poblaciones evolucionadas mostraron notables diferencias fenotípicas con respecto al aislado inicial, incluyendo cambios en la sensibilidad a estreses, crecimiento invasivo y virulencia en plantas y animales. En las poblaciones evolucionadas en medio completo se encontraron dos mutaciones recurrentes, seleccionadas en múltiples líneas de manera independiente. Los genes afectados fueron FOXG_21009 de función desconocida y FOXG_00016 (velB) que codifica un componente de Velvet, un complejo proteico regulador del desarrollo y metabolismo secundario. La caracterización de aislados monospóricos portadores de estas mutaciones, además de la generación de mutantes nulos, reveló un incremento significativo en la velocidad de crecimiento y en la capacidad de conidiación bajo las condiciones de evolución experimental. Además, se ha estudiado el papel de uno de los metabolitos secundarios regulados por Velvet, el ácido fusárico, en la infección de plantas de tomate y del hospedador invertebrado Galleria mellonella. En resumen, los resultados del presente trabajo sugieren que la plasticidad del genoma, particularmente mediante el efecto de los transposones, actúa como un importante impulsor evolutivo en F. oxysporum, y que la adaptación al huésped implica ajustes compensatorios entre distintos programas de desarrollo que promueven la infección frente a la proliferación. Fungal pathogens represent a serious threat to human health and food security. Fusarium oxysporum is a filamentous fungus that causes vascular wilt disease in more than a hundred different crops and is known for its wide ecological distribution worldwide. Although it lacks a known sexual cycle, F. oxysporum displays a striking genetic plasticity. Recognized as the first fungal multi-host pathogen model, it infects organisms from both the plant and the animal kingdom, in the latter case as an opportunistic pathogen. Its variability allows it to adapt and colonize a wide range of different niches, making it an ideal organism to study genetic mechanisms underlying adaptation. In this study we conducted experimental evolution experiments with F. oxysporum under three different conditions: plant infection, growth on minimal and on complete medium. The goal was to understand how the selective pressure exerted by these different environmental conditions drives genetic change and affects phenotype. Starting from the clonal isolate F. oxysporum f. sp lycopersici 4287, we obtained 5 independent lines evolved in each condition through ten serial passages. Genome re-sequencing of the evolved populations after the last passage revealed the presence of large segmental duplications and deletions in accessory regions of the genome, including partial loss of chromosomes. We also detected numerous transposon insertion, as well as single nucleotide polymorphisms (SNPs) and small insertions or deletions (Indels). Monitoring these events throughout the experiment by various molecular techniques allowed to establish the evolutionary dynamics within the populations as a function of the relative competitiveness of the multiple mutations propagated simultaneously. Some evolved populations showed remarkable phenotypic differences with respect to the initial isolate including changes in sensitivity to stresses, invasive growth and virulence on plant and animal hosts. In the populations evolved on complete medium, two recurrent mutations were selected independently in several lines. The affected genes were FOXG_21009 with unknown function and FOXG_00016 (velB) encoding a component of Velvet, a protein complex that regulates development and secondary metabolism. The characterization of monosporic isolates carrying these mutations, as well as the generation of deletion mutants, revealed a significant increase in growth speed and conidiation capacity under the conditions of experimental evolution. In addition, we analyzed the role of fusaric acid, one of the secondary metabolites regulated by Velvet, during infection of tomato plants and of the invertebrate host Galleria mellonella. In summary, the results of this work suggest that genome plasticity, in particular associated with transposons, acts as a potent evolutionary driver in F. oxysporum, and that host adaptation implies trade-offs between different development programs promoting infection versus proliferation.

Lentil (Lens culinaris Medikus subsp. culinaris) is one of the most important cool season food legumes. World production of lentil is estimated at 4.95 million tons from an estimated 4.34 million ha with an average yield of 1140 Kg/ha. Lentil production is threatened by biotic and abiotic stresses. One of the most destructive diseases, especially in the warm and drought conditions, is wilt disease, caused Fusarium oxysporum f.sp. lentis (Fol). Under favorable conditions this disease can cause the complete loss of the crop. The most effective, economical and environmentally friendly method to control the disease is the use of resistant cultivars. In this thesis we studied different aspects of this disease. In chapter one, 196 Spanish lentil landrace accessions were evaluated for resistance to this disease under controlled conditions using a detailed disease scoring system. Resistant accessions were further screened in the adult plant stage under naturally infested field conditions in Bileh-Savar, Iran. This study identified twelve accessions showing good levels of resistance to the disease under controlled and field conditions which could be exploited in breeding programs. In chapter two the mechanisms of resistance acting against two different pathotypes of FOL were studied in six lentil accessions showing different levels of resistance. After inoculation with patothype 1, a lower number of colonies were recovered from roots of the resistant accessions compared to the susceptible check. In the inoculation with patothype two no differences between accessions were observed for this trait. At the hystological level, qualitative resistance was detected in accession BGE019696 inoculated with pathotype 1. Thus, cells with condensed areas in the cytoplasm were observed. Furthermore, the presence of phenolic compounds was observed for this accession × pathotype interaction. This phenomenon was not observed in any other interaction. In chapter 3 the pathogenic variability of FOL was studied. Although differences in aggressiveness have been observed between FOL isolates, the presence of pathotypes has not been reported in this pathogen. The objective of chapter 3 was to check for the presence of pathotypes in FOL. As a first step to check for the presence of isolate × genotype interaction in Fol-lentil pathosysthem, 28 resistant lentil accessions were inoculated by six Fol isolates with different geographic origins. A significant isolate×genotype interaction was detected. According to this differential reaction, four accessions were selected to analyse the virulence patterns in a collection of 52 Fol isolates from Iran, Syria and Algeria. Seven different patterns of virulence were identified within this population. In chapter four the genetic structure of the Fol collection described above was analyzed using twelve SSR markers. Eight of these SSRs were developed in this thesis. AMOVA showed that there is a high molecular variation both within regions and among regions, differing Iranian populations from non-Iranian populations. Additionally, STRUCTURE and Fitch-Margoliash analysis identified two ancestral lineages, one present in all regions and the other present only in Iran. No significant relationship was found between phylogenic groups and virulence patterns. Las lentejas (Lens culinaris) son una de las leguminosas más importantes a nivel mundial. La producción mundial de lentejas se estima en 4,5 millones de toneladas, con una superficie cultivada 4,25 ha. El rendimiento de este cultivo se ve severamente afectado por diversas enfermedades, entre las que destaca la fusariosis vascular, causada por Fusarium oxysporum f. sp. lentis (FOL). Este patógeno puede causar la pérdida total de la cosecha en condiciones favorables para el desarrollo de la enfermedad. La resistencia genética se muestra como el método más efectivo, económico y respetuoso con el medio ambiente para controlar la enfermedad. En el primer capítulo de esta tesis se evaluó, en condiciones controladas, la respuesta a FOL de 196 variedades locales españolas utilizando un método detallado de evaluación desarrollado en esta tesis. Aquellas entradas que resultaron resistentes fueron también evaluadas en condiciones de campo en Bileh- Savar, Iran. Como resultado se han identificado 12 entradas de lenteja que muestran buenos niveles de resistencia tanto en condiciones controladas como en campo y que pueden ser de gran utilidad como fuentes de resistencia en programas de mejora. En el capítulo 2 se estudiaron los mecanismos de resistencia que actúan frente a dos patotipos de FOL en seis entradas de lenteja con distintos niveles de resistencia. Tras inocular con el patotipo 1, de las raíces de líneas resistentes se rescataron un menor número de colonias que en el testigo susceptible. En la inoculación con el patotipo 2 no se observaron diferencias entre líneas para este parámetro. A nivel celular se detectaron mecanismos cualitativos de resistencia en la entrada BGE019696 inoculada con el patotipo 1. Así, se observaron en esta línea células que mostraron áreas condensadas en su citoplasma. Además, en esta combinación entrada-patotipo se observó la presencia de compuestos fenólicos. Este fenómeno no se detectó en el resto de las interacciones. En el capítulo 3 se analizó la variabilidad patogénica FOL. Aunque se han descrito diferencias en agresividad entre aislados de FOL (Belabid y Fortas, 2002), hasta ahora no se habían descrito patotipos. El objetivo de este capítulo fue el estudiar si existen patotipos en FOL. Como un primer paso, para testar la existencia de interacciónes aislado x genotipo en el patosistema lenteja-FOL, se inocularon 28 genotipos de lenteja descritos como resistentes con 6 aislados del FOL de distintos orígenes geográficos. Los resultados mostraron una significativa interacción aislado x genotipo. En función de los resultados obtenidos se seleccionaron cuatro entradas de lenteja que mostraron una reacción diferencial frente a los distintos aislados para caracterizar el patrón de virulencia de una colección de 52 aislados de FOL provenientes de Iran, Siria y Argelia. Como resultado se identificaron 7 patotipos con distinto patrón de virulencia. En el capítulo 4 se estudio, utilizando doce marcadores SSR, la estructura y variabilidad genética de la colección de aislados de FOL descrita en el capítulo 3. Los análisis AMOVA mostraron que había una gran variación genética tanto dentro de regiones como entre regiones, difiriendo las poblaciones de Irán del resto de poblaciones. Por otro lado los análisis realizados con el programa STRUCTURE y el árbol de Fitch-Margoliash mostraron la existencia de dos líneas ancestrales en FOL, una que sólo estaba presente en Irán y otra que estaba distribuida por todas las regiones estudiadas. No se encontró relación entre los grupos filogenéticos y los patotipos.

El pH juega un papel clave en el control de la patogénesis fúngica. En el patógeno del suelo Fusarium oxysporum, causante de la marchitez vascular, el pH actúa como regulador maestro de Fmk1, una MAPK altamente conservada, esencial para el crecimiento invasivo y patogénesis en raíces de tomate. Estudios previos demostraron que el pH también regula las MAPKs Mpk1 y Hog1, de manera opuesta a Fmk1. Sin embargo, se desconocen los mecanismos moleculares de la respuesta al pH. En el presente trabajo se ha investigado, si el pH del medio afecta las rutas MAPK a través de un cambio en el pH intracelular (pHi). Se ha determinado que el pHi funciona como una señal para la regulación de las rutas MAPK en F. oxysporum. El tratamiento de las células con diethylstilbestrol (DES), un inhibidor especifico de Pma1, la principal bomba de protones de la membrana plasmática, conlleva una rápida y sostenida bajada del pHi, acompañada de una rápida y transitoria activación de las MAPKs Mpk1 y Hog1, y la inactivación de Fmk1. Esta respuesta esta conservada en la levadura panadera Saccharomyces cerevisiae, lo que indica que está conservado en hongos. Por ello se ha utilizado la levadura como modelo para investigar el mecanismo de activación de Mpk1 por acidificación del pHi. Se identificaron: una ruta alternativa de la respuesta a stress de pared, mediada por la quinasa Ypk1/2; la rama Sln1 de la ruta Hog1; y la MAPK fosfatasa Msg5 cuya abundancia es afectada por el pHi. En segundo lugar, un escrutinio realizado con mutantes knockout sensibles a ácido identificó un gran número de proteínas candidatas implicadas en la activación de Mpk1 inducida por DES. Muchos de ellos poseen ortólogos en F. oxysporum. Las funciones celulares más relevantes en este proceso son tráfico celular, homeostasis de iones (principalmente V-ATPase) y metabolismo de lípidos. Los resultados revelan que el pHi es un nuevo mecanismo de regulación de MAPKs en hongos, y sugieren que existe una compleja interacción entre rutas de señalización y funciones celulares en la regulación de este proceso. pH is as a key player in the control of fungal pathogenicity. In the soilborne vascular wilt fungus Fusarium oxysporum, ambient pH acts as a master switch for regulation of the conserved Fmk1 MAPK cascade, essential for invasive growth and pathogenicity toward tomato roots. Previous studies indicated that pH also modulates the activity of the two other MAPKs, Mpk1 and Hog1, in an opposite way to Fmk1. The molecular events underlying the pH response are currently unknown. Here we investigated whether ambient pH modulates MAPK signaling by affecting the intracellular pH (pHi). We found that pHi acts as a signal regulating MAPK activity in F. oxysporum. Diethylstilbestrol (DES), a specific inhibitor of the plasma membrane H+-ATPase Pma1, induced a rapid and sustained decrease of pHi accompanied by rapid and transitory phosphorylation of the MAPKs Mpk1 and Hog1, and inactivation of Fmk1. Analogously, pHi also regulates MAPK signaling in the budding yeast Saccharomyces cerevisiae, suggesting that this mechanism is conserved in fungi. We initially investigated the mechanism of pHi-induced Mpk1 activation in the yeast model, using a candidate gene approach. Our studies revealed three potential mechanisms: an alternative branch of the canonical cell wall integrity pathway mediated by the Ypk1/2 kinase; the Sln1 branch of the Hog1 cascade; and the dual specificity MAPK phosphatase Msg5 whose abundance is affected by pHi. Moreover, a screen with a subset of acid-sensitive yeast mutants identified a number of candidates affected in DES-triggered Mpk1 activation, many of which have predicted orthologues in F. oxysporum. These candidate proteins have conserved cellular functions including cellular trafficking, ion homeostasis (mainly V-ATPase function) and lipid metabolism. Our results discover pHi as a new mechanism for MAPK signaling in fungi and reveal a complex interplay between signaling pathways and cellular functions in the regulation of this process.

Lentil (Lens culinaris Medikus subsp. culinaris) is one of the most important cool season food legumes. World production of lentil is estimated at 4.95 million tons from an estimated 4.34 million ha with an average yield of 1140 Kg/ha. Lentil production is threatened by biotic and abiotic stresses. One of the most destructive diseases, especially in the warm and drought conditions, is wilt disease, caused Fusarium oxysporum f.sp. lentis (Fol). Under favorable conditions this disease can cause the complete loss of the crop. The most effective, economical and environmentally friendly method to control the disease is the use of resistant cultivars. In this thesis we studied different aspects of this disease. In chapter one, 196 Spanish lentil landrace accessions were evaluated for resistance to this disease under controlled conditions using a detailed disease scoring system. Resistant accessions were further screened in the adult plant stage under naturally infested field conditions in Bileh-Savar, Iran. This study identified twelve accessions showing good levels of resistance to the disease under controlled and field conditions which could be exploited in breeding programs. In chapter two the mechanisms of resistance acting against two different pathotypes of FOL were studied in six lentil accessions showing different levels of resistance. After inoculation with patothype 1, a lower number of colonies were recovered from roots of the resistant accessions compared to the susceptible check. In the inoculation with patothype two no differences between accessions were observed for this trait. At the hystological level, qualitative resistance was detected in accession BGE019696 inoculated with pathotype 1. Thus, cells with condensed areas in the cytoplasm were observed. Furthermore, the presence of phenolic compounds was observed for this accession × pathotype interaction. This phenomenon was not observed in any other interaction. In chapter 3 the pathogenic variability of FOL was studied. Although differences in aggressiveness have been observed between FOL isolates, the presence of pathotypes has not been reported in this pathogen. The objective of chapter 3 was to check for the presence of pathotypes in FOL. As a first step to check for the presence of isolate × genotype interaction in Fol-lentil pathosysthem, 28 resistant lentil accessions were inoculated by six Fol isolates with different geographic origins. A significant isolate×genotype interaction was detected. According to this differential reaction, four accessions were selected to analyse the virulence patterns in a collection of 52 Fol isolates from Iran, Syria and Algeria. Seven different patterns of virulence were identified within this population. In chapter four the genetic structure of the Fol collection described above was analyzed using twelve SSR markers. Eight of these SSRs were developed in this thesis. AMOVA showed that there is a high molecular variation both within regions and among regions, differing Iranian populations from non-Iranian populations. Additionally, STRUCTURE and Fitch-Margoliash analysis identified two ancestral lineages, one present in all regions and the other present only in Iran. No significant relationship was found between phylogenic groups and virulence patterns. Las lentejas (Lens culinaris) son una de las leguminosas más importantes a nivel mundial. La producción mundial de lentejas se estima en 4,5 millones de toneladas, con una superficie cultivada 4,25 ha. El rendimiento de este cultivo se ve severamente afectado por diversas enfermedades, entre las que destaca la fusariosis vascular, causada por Fusarium oxysporum f. sp. lentis (FOL). Este patógeno puede causar la pérdida total de la cosecha en condiciones favorables para el desarrollo de la enfermedad. La resistencia genética se muestra como el método más efectivo, económico y respetuoso con el medio ambiente para controlar la enfermedad. En el primer capítulo de esta tesis se evaluó, en condiciones controladas, la respuesta a FOL de 196 variedades locales españolas utilizando un método detallado de evaluación desarrollado en esta tesis. Aquellas entradas que resultaron resistentes fueron también evaluadas en condiciones de campo en Bileh- Savar, Iran. Como resultado se han identificado 12 entradas de lenteja que muestran buenos niveles de resistencia tanto en condiciones controladas como en campo y que pueden ser de gran utilidad como fuentes de resistencia en programas de mejora. En el capítulo 2 se estudiaron los mecanismos de resistencia que actúan frente a dos patotipos de FOL en seis entradas de lenteja con distintos niveles de resistencia. Tras inocular con el patotipo 1, de las raíces de líneas resistentes se rescataron un menor número de colonias que en el testigo susceptible. En la inoculación con el patotipo 2 no se observaron diferencias entre líneas para este parámetro. A nivel celular se detectaron mecanismos cualitativos de resistencia en la entrada BGE019696 inoculada con el patotipo 1. Así, se observaron en esta línea células que mostraron áreas condensadas en su citoplasma. Además, en esta combinación entrada-patotipo se observó la presencia de compuestos fenólicos. Este fenómeno no se detectó en el resto de las interacciones. En el capítulo 3 se analizó la variabilidad patogénica FOL. Aunque se han descrito diferencias en agresividad entre aislados de FOL (Belabid y Fortas, 2002), hasta ahora no se habían descrito patotipos. El objetivo de este capítulo fue el estudiar si existen patotipos en FOL. Como un primer paso, para testar la existencia de interacciónes aislado x genotipo en el patosistema lenteja-FOL, se inocularon 28 genotipos de lenteja descritos como resistentes con 6 aislados del FOL de distintos orígenes geográficos. Los resultados mostraron una significativa interacción aislado x genotipo. En función de los resultados obtenidos se seleccionaron cuatro entradas de lenteja que mostraron una reacción diferencial frente a los distintos aislados para caracterizar el patrón de virulencia de una colección de 52 aislados de FOL provenientes de Iran, Siria y Argelia. Como resultado se identificaron 7 patotipos con distinto patrón de virulencia. En el capítulo 4 se estudio, utilizando doce marcadores SSR, la estructura y variabilidad genética de la colección de aislados de FOL descrita en el capítulo 3. Los análisis AMOVA mostraron que había una gran variación genética tanto dentro de regiones como entre regiones, difiriendo las poblaciones de Irán del resto de poblaciones. Por otro lado los análisis realizados con el programa STRUCTURE y el árbol de Fitch-Margoliash mostraron la existencia de dos líneas ancestrales en FOL, una que sólo estaba presente en Irán y otra que estaba distribuida por todas las regiones estudiadas. No se encontró relación entre los grupos filogenéticos y los patotipos.

This study aimed to assess the efficacy of O. vulgare L. and O. majorana L. essential oil in inhibiting the growth and survival of potentially pathogenic fungal strains and also sought to evaluate the possible mechanisms involved in the establishment of the antifungal property of the tested essential oils through assays of osmotic stability and morphogenesis. Test strains included in this study were Candida albicans ATCC 7645, C. tropicalis LM-14, C. krusei LM-09, Cryptococcus neoformans FGF-5, Aspergillus flavus LM-02, A. fumigatus IPP-21, T. rubrum ATCC 28184, T. mentagrophytes LM-64, Microsporum gypseum ATCC 184, M. canis LM-36 and Cladosporium herbarium ATCC 26362. O. vulgare essential oil presented a MIC value of 80 µL/mL, while for O. majorana this was 160 µL/mL. C. krusei LM-09 was the only strain resistant to all assayed concentrations of both essential oils. O. vulgare and O. majorana essential oil at their MIC values provided a cidal effect against C. albicans ATCC 7645 after 4 h of exposure. O. vulgare essential oil at 80 µL/mL exhibited 100 % inhibition of the radial mycelia growth of T. rubrum ATCC 28184 and M. canis LM-36 for 14 days. Assayed fungus strain protected by sorbitol (osmo-protectant agent) grew in media containing higher concentrations of O. vulgare and O. majorana essential oil in comparison to media without sorbitol, suggesting some specificity of these essential oils for targeting cell wall in the fungi cell. Main morphological changes observed under light microscopy provided by the essential oil of O. vulgare in A. flavus LM-02 were decreased conidiation, leakage of cytoplasm, loss of pigmentation and disrupted cell structure indicating fungal wall degeneration. These results suggest that essential oils from Origanum could be regarded as a potential antifungal compound for controlling the growth of pathogen fungi and the occurrence of mycoses.O objetivo deste estudo foi observar a eficácia do óleo essencial de O. vulgare L. e O. majorana L. na inibição do crescimento e sobrevivência de cepas de fungos potencialmente patogênicas, bem como avaliar os possíveis mecanismos envolvidos no estabelecimento da propriedade antifúngica dos óleos essenciais testados através do ensaio de estabilidade osmótica e morfogênese. As cepas fúngicas utilizadas neste estudo foram Candida albicans ATCC 7645, C. tropicalis LM-14, C. krusei LM-09, Cryptococcus neoformans FGF-5, Aspergillus flavus LM-02, A. fumigatus IPP-21, T. rubrum ATCC 28184, T. mentagrophytes LM-64, Microsporum gypseum ATCC 184, M. canis LM-36 e Cladosporium herbarium ATCC 26362. O óleo essencial de O. vulgare apresentou valor de CIM de 80 µL/mL, enquanto o óleo essencial de O. majorana apresentou valor de CIM de 160 µL/mL. C. krusei LM-09 apresentou-se como a única cepa resistente a todas as concentrações ensaiadas de ambos os óleos essenciais. Os óleos essenciais testados quando ensaiadas em seu valor de CIM causaram um efeito fungicida contra C. albicans ATCC 7645 após 4 h de exposição. O óleo essencial de O. vulgare na concentração de 80 µL/mL exibiu uma total inibição do crescimento micelial radial de T. rubrum ATCC 28184 e M. canis LM-36 ao longo de 14 dias. As cepas fúngicas ensaiadas quando tratadas com sorbitol (agente osmo-protetor) foram capazes de crescer em meio adicionado de mais altas concentrações dos óleos essenciais quando comparados ao meio não adicionado de sorbitol, sugerindo especificidade destes produtos a parede celular como alvo na célula fúngica. As principais alterações causadas pelo óleo essencial de O. vulgare sobre a morfologia de A. flavus LM-02 foram diminuída conidiação, perda de citoplasma, perda de pigmentação e ruptura da estrutura celular indicando degeneração da parede celular fúngica. Estes resultados sugerem que óleos essenciais de espécies de Origanum poderiam ser considerados como potenciais antifúngicos para o controle de fungos patógenos e ocorrência de micoses.

Premio extraordinario de Trabajo Fin de Máster curso 2013-2014. Molecular and Cellular, Biotechnology and Genetics. El pH extracelular juega un papel clave en los niveles de fosforilación de las MAP quinasas de Fusarium oxysporum. Además, existen evidencias significativas de que la virulencia de distintos patógenos fúngicos se ve alterada directamente por el pH extracelular. Actualmente se desconoce como el pH extracelular afecta al pH intracelular. En este trabajo, hemos hecho uso de una proteína fluorescente sensible al pH, la pHluorin. Dicha proteína posee la misma estructura que la GFP, pero con modificaciones que permiten medir el pH en el interior celular como valor radiométrico a dos longitudes de excitación y una de emisión. Esta proteína ha sido ampliamente utilizada en la levadura Saccharomyces cerevisiae. El objetivo de este trabajo ha sido la construcción de cepas de F. oxysporum que expresan de forma constitutiva el gen de la pHluorin, y la optimización de las condiciones experimentales para la medida del pH intracelular. Una vez optimizada la metodología, se identificaron cambios significativos en el pH intracelular cuando la célula fue sometida a cambios drásticos de pH extracelular. Cuando las germínulas se sometieron a pH 9 se observó una subida rápida, seguida por una bajada gradual hasta alcanzar el valor homeostático entre 5.5 y 6.5. Por el contrario, cuando las germínulas se sometieron a pH 4 se detectó una bajada seguida por una subida muy lenta, siendo aún desconocido el mecanismo por el cual la homeostasis de pH es llevada a cabo. Además se observó una acidificación en presencia de glucosa, de forma opuesta a lo anteriormente descrito en levadura. Esta acidificación puede deberse a la entrada de glucosa en el interior celular, mediada por un sinporte con protones. Futuros experimentos serán necesarios para determinar los mecanismos de regulación de la homeostasis del pH intracelular cuando las germínulas se someten a pH 4 y pH 9 y su papel en la señalización y la virulencia de este patógeno.

Pesquisas têm demonstrado que os plantios florestais podem acelerar os processos sucessionais e ecológicos da regeneração natural e recuperação de áreas degradadas, reabilitando o meio ambiente, conservando o solo e produzindo madeira, e assim, contribuindo para redução dos desmatamentos das florestas nativas. No entanto, pouco se conhece sobre os problemas fitossanitários que ocorrem nas espécies estabelecidas nesses plantios. Neste sentido, objetivando conhecer as doenças que possam afetar o desenvolvimento das plantas, em 2005 foi conduzido um levantamento pioneiro em plantios florestais demonstrativos nos municípios de Manaus e Presidente Figueiredo – AM, onde foram identificados dez fungos patogênicos causadores de manchas foliares distintas e quatro fungos causadores de fuligens, encontrados nas espécies florestais estudadas: Cylindrocladium sp. e Meliola sp. em andiroba; Sclerotium coffeicola, Colletotrichum gloeosporioides e Meliola sp. em mogno; Cercospora sp., Rhizoctonia sp., Pseudobeltrania cedrelae e Meliola sp. em cedro; Cercospora bertholletia e Yamamotoa sp. em castanheira-do-brasil; Erythrogloeum hymenaeae, Colletotrichum sp., Pestalotiopsis sp., Meliola sp., Amazonia sp. e Asterina sp. em jatobá e Rhizoctonia sp. em pau-de-balsa. Testes de patogenicidade e reisolamento dos patógenos foram conduzidos em casa de vegetação e laboratórios do INPA, cumprindo os Postulados de Koch. Nos plantios florestais a severidade das doenças encontradas foi de maneira geral baixa, no período em que foram observadas. Jatobá e cedro foram as espécies florestais que apresentaram maior incidência de doenças. Não foram encontrados registros anteriores sobre a ocorrência de Meliola sp. em mogno, Cercospora sp., Rhizoctonia sp. e Meliola sp.em cedro, Pestalotiopsis sp., Meliola sp., Amazonia sp. e Asterina sp. em jatobá e Rhizoctonia sp. em pau-de-balsa. Este é o primeiro registro destes patógenos atacando estas espécies. Researches has shown that Forest plantations could accelerate the successive and ecologic processes of natural regeneration and degraded reas recovering, reestablishing the environment, conserving the soil and producing timber, thus contributing for deforestation reduction of native forests. Nevertheless, very little is known about the phytosanitary problems that happen in the species already established on those plantations. On this sense, it is aiming at to know the diseases that could affect the developing of plants, in 2005, it was conducted a pioneer survey at demostrative forest plantations areas in Manaus and Presidente Figueiredo - AM counties, where there were identified ten patogenous fungus causative of diferent spot – leaf and four fungus causative of soots found in the studied forest species: Cylindrocladium sp. and Meliola sp. in crab - wood (Carapa guianensis); Colletotrichum gloeosporioides, Sclerotium coffeicola and Meliola sp. in mahogany (Swietenia macrophylla); Cercospora sp., Rhizoctonia sp. Pseudobeltrania cedrelae and Meliola sp. in Cedar (Cedrela odorata); Cercospora bertholletia and Yamamotoa sp. in brazilian nut tree (Bertholletia excelsa); Erythrogloeum hymenaeae, Colletotrichum sp. Pestalotiopsis sp., Meliola sp., Asterina sp. and Amazonia sp. in stinking - toe (Hymenaea courbaril) and Rhizoctonia sp. in cork – wood (Ochroma piramidale). Tests of pathogenicity and reisolation of pathogenous were conducted at Inpa's green house and laboratories as well, following the Koch Postulate. In forest plantations, the illness severity found in a general approach was low quite. During the observing period, stinking - toe and cedar were the forest species that has shown the greatest illness incidence. It hasn't been found former registers about occurrences on Meliola sp. em mahogany, Cercospora sp., Rhizoctonia sp. e Meliola sp. in Cedar, Pestalotiopsis sp., Meliola sp., Amazonia sp. e Asterina sp. in stinking – toe and Rhizoctonia sp. in cork – wood. This is the very first register of these pathogenous attaching these species. x

Hemos querido presentar en este libro una extensa información sobre las enfermedades que afectan a nuestros agrios durante la postrecolección, o sea, a lo largo de todo el período de la comercialización de los frutos. La necesidad de una publicación de esta clase, ha sido puesta a nuestra atención reiteradamente por los técnicos de los almacenes acondicionadores, de las inspecciones fitosanitarias, de las empresas fitosanitarias así como por numerosos interesados en el cultivo de los agrios. Los objetivos propuestos en este libro, como son: a) conocer e identificar cuales son las enfermedades de los agrios en España después de ser recolectados; b) diagnosticar al agente o agentes micóticos causales; c) aplicar las medidas más idóneas para su control; son amplios e importantes y pueden servir no solo de guía a los técnicos mencionados sino también a estudiantes e investigadores que se inician en la patología de los agrios. Necesidad que es muy requerida, ya que -por lo general- la patología de la postrecolección tiende a ser olvidada u omitida en los libros de texto y es, practicamente, desconocida en castellano.