DETECTION OF SPORES AND HYPHAE OF ARTWORKS’ BIODETERIOGENIC FILAMENTOUS FUNGI BY RNA-FISH

Filamentous fungi are a threat to the conservation of Cultural Heritage. Thus, detection and identification of viable filamentous fungi are crucial for applying adequate Safeguard measures. RNA-FISH protocols have been previously applied with this aim in Cultural Heritage samples. However, only hyphae detection was reported in the literature, even if spores and conidia are not only a potential risk to Cultural Heritage but can also be harmful for the health of visitors, curators and restorers. Thus, the aim of this work was to evaluate various permeabilizing strategies for their application in the detection of spores/conidia and hyphae of artworks’ biodeteriogenic filamentous fungi by RNA-FISH. Besides of this, the influence of cell aging on the success of the technique and on the development of fungal autofluorescence (that could hamper the RNA-FISH signal detection) were also investigated. Five common biodeteriogenic filamentous fungi species isolated from biodegradated artworks were used as biological model: Aspergillus niger, Cladosporium sp, Fusarium sp, Penicillium sp. and Exophialia sp. Fungal autofluorescence was only detected in cells harvested from Fusarium sp, and Exophialia sp. old cultures, being aging-dependent. However, it was weak enough to allow autofluorescence/RNA-FISH signals distinction. Thus, autofluorescence was not a limitation for the application of RNA-FISH for detection of the taxa investigated. All the permeabilization strategies tested allowed to detect fungal cells from young cultures by RNA-FISH. However, only the combination of paraformaldehyde with Triton X-100 allowed the detection of conidia/spores and hyphae of old filamentous fungi. All the permeabilization strategies failed in the Aspergillus niger conidia/spores staining, which are known to be particularly difficult to permeabilize. But, even in spite of this, the application of this permeabilization method increased the analytical potential of RNA FISH in Cultural Heritage biodeterioration. Whereas much work is required to validate this RNA-FISH approach for its application in real samples from Cultural Heritage it could represent an important advance for the detection, not only of hyphae but also of spores and conidia of various filamentous fungi taxa by RNA-FISH.

Isolamento e seleção de fungos filamentosos resistentes a atrazina.

Embora a toxicidade de alguns herbicidas tenha sido explorada em profundidade, seu destino no ambiente e sua transformação não são bem compreendidos. a fim de melhor avaliar os impactos da atrazina, e crucial que informações sobre sua biodegradação seja explorada. De solos agrícolas com repetidas aplicações deste herbicida, como também de solos enriquecidos, foram isolados 29 fungos com resistência a altas concentrações do produto 2.000 a 7.000 ppm. Destes fungos, 9 (nove) dos gêneros Penicillium sp. e Eupenicillium sp. mostraram-se capazes de degradação que varia de 26% a 92% dentro de 21 dias, em geral. Os fungos foram cultivados em meio de cultura liquido e incubados no escuro a 28.o C, sob agitação (180 rpm). A extração foi feita com acetato de etila e o consumo da atrazina, pelos fungos, avaliado através de Cromatografia Gasosa com Detecção Termoionica específica a nitrogênio e fósforo.

Fontes Fosfatadas, Ácidos Húmicos e Fungos Solubilizadores de Fosfato em Trigo

Este trabalho objetivou avaliar o efeito de fontes fosfatadas, ácidos húmicos e fungos solubilizadores de fosfato em trigo cultivado em solo de Cerrado. O experimento foi instalado em delineamento inteiramente casualizado com 7 tratamentos: 1) ácidos húmicos (30 kg ha-1); 2) ácidos húmicos + fosfato de Arad (800 kg ha-1); 3) ácidos húmicos + pó de basalto (526 kg ha-1); 4) fosfato de Arad; 5) pó de basalto; 6) FSF (108 UFC mL-1), 7) testemunha e 4 repetições. As espécies de fungo usadas foram: um isolado de Penicillium sp e um de Aspergillus sp. A combinação de fosfato de Arad e ácidos húmicos incrementou o desenvolvimento de trigo. A inoculação de FSF não se mostrou efetiva para beneficiar o desenvolvimento de trigo.

Comparison of two marine sponge-associated Penicillium strains DQ25 and SC10: differences in secondary metabolites and their bioactivities

Two strains of Penicillium, DQ25 and SC10, isolated from marine sponge Haliclona angulata (Bowerbank) and Hymeniacidon sp. respectively, were subjected to stationary cultivation under GYP medium for 30 days. The fermentation extracts were undergone bioactivities assays against human pathogens, phytopathogenic fungi and brine shrimp (Artemia salina). Bioassays-guided compounds isolation was performed by Silica gel columns and Sephadex LH-20 chromatography. Spectroscopic methods were used to structures elucidation of the compounds. Results showed the activities of secondary metabolites of strain DQ25 were generally stronger than that of strain SC10. Major bioactive molecules isolated from strain DQ25 were a 1,4-naphthoquinone derivative and an unidentified alkaloid. The two components were not isolated from the extract of strain SC10. ITS sequences revealed that these two species have the greatest similarity with Penicillium vinaceum and Penicillium granulatum respectively.

Use of Experimental Design for the Optimization of the Production of New Secondary Metabolites by Two Penicillium Species

A fractional factorial design approach has been used to enhance secondary metabolite production by two Penicillium strains. The method was initially used to improve the production of bioactive extracts as a whole and subsequently to optimize the production of particular bioactive metabolites. Enhancements of over 500% in secondary metabolite production were observed for both P. oxalicum and P. citrinum. Two new alkaloids, citrinalins A (5) and B (6), were isolated and identified from P. citrinum cultures optimized for production of minor metabolites.

Fungos anamorfos do solo da região dos lagos no Município de Santa Gertrudes, SP, Brasil

O solo do pólo cerâmico no município de Santa Gertrudes, SP, tem sido poluído há décadas por diversos elementos químicos, principalmente chumbo e zinco. Foram realizadas quatro coletas de amostras de solo, duas durante a época chuvosa e duas na seca, em cinco locais, de novembro de 2002 a junho de 2003, determinando-se temperatura, pH, teores de chumbo e zinco e a umidade do solo. Os fungos foram isolados pelo método de Warcup, modificado pelo preparo de suspensões aquosas de solo (1:10) e aplicação de 1 cm³ das suspensões sobre malte agar (2%), adicionado de Zn(NO3)2 e Pb(NO3)2 em concentrações crescentes: 0, 100, 200, 500 e 1.000 mg dm-3. Após cinco dias de incubação a 25 ºC, as colônias foram purificadas e identificadas. Foram obtidos 70 táxons de fungos anamorfos, com 70% de similaridade entre as micotas obtidas nos meios com os dois metais. Foram isolados 43 táxons nos meios de cultura com Pb(NO3)2, com predominância deles nas concentrações mais elevadas (500 a 1.000 mg dm-3). Foram obtidos 63 táxons nos meios com Zn(NO3)2, principalmente nas concentrações moderada e elevada (200 e 500 mg dm-3). Prevaleceram espécies de Trichoderma, de Penicillium e diversos fungos que são encontrados associados a substratos vegetais em decomposição. A tendência de se obter número elevado de táxons em meios de cultura com concentrações moderadas a elevadas de Zn e Pb pode ser justificada pela existência de bem adaptada e competitiva micota do solo, caracterizada por elevada capacidade de tolerância aos metais e eficiente habilidade sapróbia competitiva.

Microbiota fúngica de um prédio com laboratórios da região de Araraquara-SP

From September 2000 to January 2001, airborne fungi were isolated from the building of the Clinical Analyses laboratories, including its didactic and research rooms, in Araraquara São Paulo State, Brazil, by using Andersen, MAS-100® (MERCK) machine, with Sabouraud chloramphenicol medium. After 5 days of incubation at 25°C, the colonies of the fungi were counted, resulting in the identification of 21 taxa. Cladophialophora spp. was the most isolated in internal and external environments as well, followed by Penicillium spp. and Mycelia spp. In accordance with the resolution n° 9, January 2003 (ANVISA), fungi considered unacceptable were found in nine internal environments and one of these presented the amount of fungi above of the acceptable limit. Among the obtained fungi, at least 16 taxa were reported as opportunistic, nine were related to plant diseases and seven were associated to allergy problems.

Controle de Guignardia citricarpa e Penicillium digitatum em laranja com óleos essenciais e agentes de biocontrole

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Marine Fungi Aspergillus sydowii and Trichoderma sp Catalyze the Hydrolysis of Benzyl Glycidyl Ether

Whole cells of the marine fungi Aspergillus sydowii Gc12, Penicillium raistrickii Ce16, P. miczynskii Gc5, and Trichoderma sp. Gc1, isolated from marine sponges of the South Atlantic Ocean (Brazil), have been screened for the enzymatic resolution of (+/-)-2-(benzyloxymethyl)oxirane (benzyl glycidyl ether; 1). Whole cells of A. sydowii Gc12 catalyzed the enzymatic hydrolysis of (R,S)-1 to yield (R)-1 with an enantiomeric excess (ee) of 24-46% and 3-(benzyloxy)propane-1,2-diol (2) with ee values < 10%. In contrast, whole cells of Trichoderma sp. Gc1 afforded (S)-1 with ee values up to 60% and yields up to 39%, together with (R)-2 in 25% yield and an ee of 32%. This is the first published example of the hydrolysis of 1 by whole cells of marine fungi isolated from the South Atlantic Ocean. The hydrolases from the two studied fungi exhibited complementary regioselectivity in opening the epoxide ring of racemic 1, with those of A. sydowii Gc12 showing an (S) preference and those of Trichoderma sp. Gc1 presenting an (R) preference for the substrate.

Enzimi, acidi organici ed altri metaboliti coinvolti nella patogenesi di Penicillium spp

Blue mould caused by Penicillium expansum Link is one of the most destructive rot of pome fruit in all growing areas (Snowdon, 1990; Jones and Aldwinckle, 1991; Tonini,1996) In the past, Penicillium rot has been controlled by fungicide postharvest treatment mainly by thiabendazole (TBZ) and benomyl (Hardenburg and Spalding, 1972), but their intense use produced the appearance of resistant strains with a great reduction of their activity The aims of the present study were to characterize the isolates of Pencillium sp causing blue mold on pear in Italy by physiological and biochemical parameters. In particular differencing also the behavior of isolates to relationship with sensitivity or resistance to TBZ treatments. We have examined the early stage of infection in relation to enzyme activity, local modulation of pH, production of organic acids, and to secondary metabolism of pathogen. The results described here confirm that the majority of P. expansum isolates from pears packing houses are resistant to TBZ, Among the TBZ-resistant isolates scored in this work, different isolates (RR) showed higher percentage of conidial germination on TBZ-amended medium compared to non amended medium. This may indicate a stimulatory effect of TBZ on conidial germination. Therefore TBZ treatments are not only ineffective for controlling P. expansum, but they may also increase the severity of blue mould on fruits. In the absence of fungicide, isolates showed a significant difference for infection severity, R and RR isolates are characterized by higher pathogenic fitness on fruits, producing larger lesions than S isolates. These data are supported by the study with laboratory-induced resistant isolates, which shows the lack of correlation between TBZ resistance and osmotic sensitivity, and highlights the association between TBZ resistance and infection severity (Baraldi et al 2003). Enzymatic screening gave a positive reaction to esterase, urease, pectinase activity, in addition, the pathogen is able to synthesize a complex enzyme act to degrade the main components of the cell wall especially pectin and cellulose. Isolated sensitive and resistant are characterized by a good activity of pectinase, especially from poligactoronase, which, as already reported by several studies (D'hallewin et al, 2004; Prusky et al, 2004), are the basis of degradative process of cell wall. Also, although the measure was minor also highlighted some activities of cellulase, but even note in the production of this kind of cellulase and hemicellulase P. Expansum were not targeted, studies have found no other source of information in this regard. Twenty isolates of Penicillium expansum, were tested in vitro ad in vivo for acid production ability and pH drop. We have found that modulation of pH and the organic acids extrusion were influence to various parameter:  Initial pH: in general, the greatest reduction of pH was observed in isolates grown at pH 7, except for four isolates that maintained the pH of the medium close to 7, the others significantly decreased the pH, ranging from 5.5 to 4.1.. In extreme acid condition (pH 3,0) growth and modulation of pH is most lower respect optimal condition (pH 5,0). Also isolates R and RR have showed a greater adaptation to environmental condition more than isolates S.  Time: although the acidification continues for some days, PH modulation is strongest in early hours (48-72 hours)of inoculation process. Time also affects the quality of organic acids, for example in vitro results showed an initial abundant production of succinc acid, followed to important production of galacturoinc acid.  Substrates: there are many differences for the type of acids produced in vitro and in vivo. Results showed in vivo an abundant production of galacturonic, malic, and citric acids and some unknown organic acids in smaller concentrations. Secondary metabolite analysis revealed intra-specific differences, and patulin was found in all isolates, but most significant reduction was observed between in vitro and in vivo samples. There was no correlation between the concentration of patulin, and the percentage of infected fruits, but sample with a lower infection severity of rotten area than the others, showed a significantly lower mycotoxin concentration than samples with a higher lesion diameter of rotten area. Beyond of patulin was detected the presence of another secondary metabolite, penitrem A.

Mejora de la eficacia de Penicillium oxalicum como agente de biocontrol en enfermedades de plantas hortícolas

Actualmente, la reducción de materias activas (UE) y la implantación de la nueva Directiva comunitaria 2009/128/ que establece el marco de actuación para conseguir un uso sostenible de los plaguicidas químicos y la preferencia de uso de métodos biológicos, físicos y otros no químicos, obliga a buscar métodos de control menos perjudiciales para el medio ambiente. El control biológico (CB) de enfermedades vegetales empleando agentes de control biológico (ACB) se percibe como una alternativa más segura y con menor impacto ambiental, bien solos o bien como parte de una estrategia de control integrado. El aislado 212 de Penicillium oxalicum (PO212) (ATCC 201888) fue aislado originalmente de la micoflora del suelo en España y ha demostrado ser un eficaz ACB frente a la marchitez vascular del tomate. Una vez identificado y caracterizado el ACB se inició el periodo de desarrollo del mismo poniendo a punto un método de producción en masa de sus conidias. Tras lo cual se inició el proceso de formulación del ACB deshidratando las conidias para su preservación durante un período de tiempo mayor mediante lecho fluido. Finalmente, se han desarrollado algunos formulados que contienen de forma individual diferentes aditivos que han alargado su viabilidad, estabilidad y facilitado su manejo y aplicación. Sin embargo, es necesario seguir trabajando en la mejora de su eficacia de biocontrol. El primer objetivo de esta Tesis se ha centrado en el estudio de la interacción ACB-patógeno-huésped que permita la actuación de P.oxalicum en diferentes patosistemas. Uno de los primeros puntos que se abordan dentro de este objetivo es el desarrollo de nuevas FORMULACIONES del ACB que incrementen su eficacia frente a la marchitez vascular del tomate. Las conidias formuladas de PO212 se obtuvieron por la adición conjunta de distintos aditivos (mojantes, adherentes o estabilizantes) en dos momentos diferentes del proceso de producción/secado: i) antes del proceso de producción (en la bolsa de fermentación) en el momento de la inoculación de las bolsas de fermentación con conidias de PO212 o ii) antes del secado en el momento de la resuspensión de las conidias tras su centrifugación. De las 22 nuevas formulaciones desarrolladas y evaluadas en plantas de tomate en ensayos en invernadero, seis de ellas (FOR22, FOR25, FOR32, FOR35, FOR36 y FOR37) mejoran significativamente (P=0,05) el control de la marchitez vascular del tomate con respecto al obtenido con las conidias secas de P.oxalicum sin aditivos (CSPO) o con el fungicida Bavistin. Los formulados que mejoran la eficacia de las conidias secas sin aditivos son aquellos que contienen como humectantes alginato sódico en fermentación, seguido de aquellos que contienen glicerol como estabilizante en fermentación, y metil celulosa y leche desnatada como adherentes antes del secado. Además, el control de la marchitez vascular del tomate por parte de los formulados de P. oxalicum está relacionado con la fecha de inicio de la enfermedad. Otra forma de continuar mejorando la eficacia de biocontrol es mejorar la materia activa mediante la SELECCIÓN DE NUEVAS CEPAS de P. oxalicum, las cuales podrían tener diferentes niveles de eficacia. De entre las 28 nuevas cepas de P. oxalicum ensayadas en cámara de cultivo, sólo el aislado PO15 muestra el mismo nivel de eficacia que PO212 (62-67% de control) frente a la marchitez vascular del tomate en casos de alta presión de enfermedad. Mientras que, en casos de baja presión de enfermedad todas las cepas de P. oxalicum y sus mezclas demuestran ser eficaces. Finalmente, se estudia ampliar el rango de actuación de este ACB a OTROS HUÉSPEDES Y OTROS PATÓGENOS Y DIFERENTES GRADOS DE VIRULENCIA. En ensayos de eficacia de P. oxalicum frente a aislados de diferente agresividad de Verticillium spp. y Fusarium oxysporum f. sp. lycopersici en plantas de tomate en cámaras de cultivo, se demuestra que la eficacia de PO212 está negativamente correlacionada con el nivel de enfermedad causada por F. oxysporum f. sp. lycopersici pero que no hay ningún efecto diferencial en la reducción de la incidencia ni de la gravedad según la virulencia de los aislados. Sin embargo, en los ensayos realizados con V. dahliae, PO212 causa una mayor reducción de la enfermedad en las plantas inoculadas con aislados de virulencia media. La eficacia de PO212 también era mayor frente a aislados de virulencia media alta de F. oxysporum f. sp. melonis y F. oxysporum f. sp. niveum, en plantas de melón y sandía, respectivamente. En ambos huéspedes se demuestra que la dosis óptima de aplicación del ACB es de 107 conidias de PO212 g-1 de suelo de semillero, aplicada 7 días antes del trasplante. Además, entre 2 y 4 nuevas aplicaciones de PO212 a la raíces de las plantas mediante un riego al terreno de asiento mejoran la eficacia de biocontrol. La eficacia de PO212 no se limita a hongos patógenos vasculares como los citados anteriormente, sino también a otros patógenos como: Phytophthora cactorum, Globodera pallida y G. rostochiensis. PO212 reduce significativamente los síntomas (50%) causados por P. cactorum en plantas de vivero de fresa, tras la aplicación del ACB por inmersión de las raíces antes de su trasplante al suelo de viveros comerciales. Por otra parte, la exposición de los quistes de Globodera pallida y G. rostochiensis (nematodos del quiste de la patata) a las conidias de P. oxalicum, en ensayos in vitro o en microcosmos de suelo, reduce significativamente la capacidad de eclosión de los huevos. Para G. pallida esta reducción es mayor cuando se emplean exudados de raíz de patata del cv. 'Monalisa', que exudados de raíz del cv. 'Desirée'. No hay una reducción significativa en la tasa de eclosión con exudados de raíz de tomate del cv. 'San Pedro'. Para G. rostochiensis la reducción en la tasa de eclosión de los huevos se obtiene con exudados de la raíz de patata del cv. 'Desirée'. El tratamiento con P. oxalicum reduce también significativamente el número de quistes de G. pallida en macetas. Con el fin de optimizar la aplicación práctica de P. oxalicum cepa 212 como tratamiento biológico del suelo, es esencial entender cómo el entorno físico influye en la capacidad de colonización, crecimiento y supervivencia del mismo, así como el posible riesgo que puede suponer su aplicación sobre el resto de los microorganismos del ecosistema. Por ello en este segundo objetivo de esta tesis se estudia la interacción del ACB con el medio ambiente en el cual se aplica. Dentro de este objetivo se evalúa la INFLUENCIA DE LA TEMPERATURA, DISPONIBILIDAD DE AGUA Y PROPIEDADES FÍSICO-QUÍMICAS DE LOS SUELOS (POROSIDAD, TEXTURA, DENSIDAD...) SOBRE LA SUPERVIVENCIA Y EL CRECIMIENTO DE PO212 en condiciones controladas elaborando modelos que permitan predecir el impacto de cada factor ambiental en la supervivencia y crecimiento de P. oxalicum y conocer su capacidad para crecer y sobrevivir en diferentes ambientes. En las muestras de suelo se cuantifica: i) la supervivencia de Penicillium spp. usando el recuento del número de unidades formadoras de colonias en un medio de cultivo semi-selectivo y ii) el crecimiento (biomasa) de PO212 mediante PCR en tiempo real. En los resultados obtenidos se demuestra que P. oxalicum crece y sobrevive mejor en condiciones de sequía independientemente de la temperatura y del tipo de suelo. Si comparamos tipos de suelo P. oxalicum crece y sobrevive en mayor medida en suelos areno-arcillosos con un bajo contenido en materia orgánica, un mayor pH y una menor disponibilidad de fósforo y nitrógeno. La supervivencia y el crecimiento de P. oxalicum se correlaciona de forma negativa con la disponibilidad de agua y de forma positiva con el contenido de materia orgánica. Sólo la supervivencia se correlaciona también positivamente con el pH. Por otro lado se realizan ensayos en suelos de huertos comerciales con diferentes propiedades físico-químicas y diferentes condiciones ambientales para ESTUDIAR EL ESTABLECIMIENTO, SUPERVIVENCIA Y DISPERSIÓN VERTICAL Y MOVILIDAD HORIZONTAL DE PO212. P. oxalicum 212 puede persistir y sobrevivir en esos suelos al menos un año después de su liberación pero a niveles similares a los de otras especies de Penicillium indígenas presentes en los mismos suelos naturales. Además, P. oxalicum 212 muestra una dispersión vertical y movilidad horizontal muy limitada en los diferentes tipos de suelo evaluados. La introducción de P. oxalicum en un ambiente natural no sólo implica su actuación sobre el microorganismo diana, el patógeno, si no también sobre otros microorganismos indígenas. Para EVALUAR EL EFECTO DE LA APLICACIÓN DE P. oxalicum SOBRE LAS POBLACIONES FÚNGICAS INDIGENAS PRESENTES EN EL SUELO de dos huertos comerciales, se analizan mediante electroforesis en gradiente desnaturalizante de poliacrilamida (DGGE) muestras de dichos suelos a dos profundidades (5 y 10 cm) y a cuatro fechas desde la aplicación de P. oxalicum 212 (0, 75, 180 y 365 días). El análisis de la DGGE muestra que las diferencias entre las poblaciones fúngicas se deben significativamente a la fecha de muestreo y son independientes del tratamiento aplicado y de la profundidad a la que se tomen las muestras. Luego, la aplicación del ACB no afecta a la población fúngica de los dos suelos analizados. El análisis de las secuencias de la DGGE confirma los resultados anteriores y permiten identificar la presencia del ACB en los suelos. La presencia de P. oxalicum en el suelo se encuentra especialmente relacionada con factores ambientales como la humedad. Por tanto, podemos concluir que Penicillium oxalicum cepa 212 puede considerarse un óptimo Agente de Control Biológico (ACB), puesto que es ecológicamente competitivo, eficaz para combatir un amplio espectro de enfermedades y no supone un riesgo para el resto de microorganismos fúngicos no diana presentes en el lugar de aplicación. ABSTRACT Currently, reduction of active (EU) and the implementation of the new EU Directive 2009/128 which establishing the framework for action to achieve the sustainable use of chemical pesticides and preference of use of biological, physical and other non-chemical methods, forces to look for control methods less harmful to the environment. Biological control (CB) of plant diseases using biological control agents (BCA) is perceived as a safer alternative and with less environmental impact, either alone or as part of an integrated control strategy. The isolate 212 of Penicillium oxalicum (PO212) (ATCC 201888) was originally isolated from the soil mycoflora in Spain. P. oxalicum is a promising biological control agent for Fusarium wilt and other tomato diseases. Once identified and characterized the BCA, was developed a mass production method of conidia by solid-state fermentation. After determined the process of obtaining a formulated product of the BCA by drying of product by fluid-bed drying, it enables the preservation of the inoculum over a long period of time. Finally, some formulations of dried P. oxalicum conidia have been developed which contain one different additive that have improved their viability, stability and facilitated its handling and application. However, further work is needed to improve biocontrol efficacy. The first objective of this thesis has focused on the study of the interaction BCA- pathogen-host, to allow P.oxalicum to work in different pathosystems. The first point to be addressed in this objective is the development of new FORMULATIONS of BCA which increase their effectiveness against vascular wilt of tomato. PO212 conidial formulations were obtained by the joint addition of various additives (wetting agents, adhesives or stabilizers) at two different points of the production-drying process: i) to substrate in the fermentation bags before the production process, and (ii) to conidial paste obtained after production but before drying. Of the 22 new formulations developed and evaluated in tomato plants in greenhouse tests, six of them (FOR22 , FOR25 , FOR32 , FOR35 , FOR36 and FOR3) improved significantly (P = 0.05) the biocontrol efficacy against tomato wilt with respect to that obtained with dried P.oxalicum conidia without additives (CSPO) or the fungicide Bavistin. The formulations that improve the efficiency of dried conidia without additives are those containing as humectants sodium alginate in the fermentation bags, followed by those containing glycerol as a stabilizer in the fermentation bags, and methylcellulose and skimmed milk as adherents before drying. Moreover, control of vascular wilt of tomatoes by PO212 conidial formulations is related to the date of disease onset. Another way to further improve the effectiveness of biocontrol is to improve the active substance by SELECTION OF NEW STRAINS of P. oxalicum, which may have different levels of effectiveness. Of the 28 new strains of P. oxalicum tested in a culture chamber, only PO15 isolate shows the same effectiveness that PO212 (62-67 % of control) against tomato vascular wilt in cases of high disease pressure. Whereas in cases of low disease pressure all strains of P. oxalicum and its mixtures effective. Finally, we study extend the range of action of this BCA TO OTHER GUESTS AND OTHER PATHOGENS AND DIFFERENT DEGREES OF VIRULENCE. In efficacy trials of P. oxalicum against isolates of different aggressiveness of Verticillium spp. and Fusarium oxysporum f. sp. lycopersici in tomato plants in growth chambers, shows that the efficiency of PO212 is negatively correlated with the level of disease caused by F. oxysporum f. sp. lycopersici. There is not differential effect in reducing the incidence or severity depending on the virulence of isolates. However, PO212 cause a greater reduction of disease in plants inoculated with virulent isolates media of V. dahlia. PO212 efficacy was also higher against isolates of high and average virulence of F. oxysporum f. sp. melonis and F. oxysporum f. sp. niveum in melon and watermelon plants, respectively. In both hosts the optimum dose of the BCA application is 107 conidia PO212 g-1 soil, applied on seedlings 7 days before transplantation into the field. Moreover, the reapplication of PO212 (2-4 times) to the roots by irrigation into the field improve efficiency of biocontrol. The efficacy of PO212 is not limited to vascular pathogens as those mentioned above, but also other pathogens such as Oomycetes (Phytophthora cactorum) and nematodes (Globodera pallida and G. rostochiensis). PO212 significantly reduces symptoms (50 %) caused by P. cactorum in strawberry nursery plants after application of BCA by dipping the roots before transplanting to soil in commercial nurseries. Moreover, the exposure of G. pallida and G. rostochiensis cysts to the conidia of P. oxalicum, in in vitro assays or in soil microcosms significantly reduces hatchability of eggs. The reduction in the rate of G. pallida juveniles hatching was greatest when root diffusates from the `Monalisa´ potato cultivar were used, followed by root diffusates from the `Désirée´ potato cultivar. However, no significant reduction in the rate of G. pallida juveniles hatching was found when root diffusates from the ‘San Pedro” tomato cultivar were used. For G. rostochiensis reduction in the juveniles hatching is obtained from the root diffusates 'Desirée' potato cultivar. Treatment with P. oxalicum also significantly reduces the number of cysts of G. pallida in pots. In order to optimize the practical application of P. oxalicum strain 212 as a biological soil treatment, it is essential to understand how the physical environment influences the BCA colonization, survival and growth, and the possible risk that can cause its application on other microorganisms in the ecosystem of performance. Therefore, the second objective of this thesis is the interaction of the BCA with the environment in which it is applied. Within this objective is evaluated the INFLUENCE OF TEMPERATURE, WATER AVAILABILITY AND PHYSICAL-CHEMICAL PROPERTIES OF SOILS (POROSITY, TEXTURE, DENSITY...) ON SURVIVAL AND GROWTH OF PO212 under controlled conditions to develop models for predicting the environmental impact of each factor on survival and growth of P. oxalicum and to know their ability to grow and survive in different environments. Two parameters are evaluated in the soil samples: i) the survival of Penicillium spp. by counting the number of colony forming units in semi-selective medium and ii) growth (biomass) of PO212 by real-time PCR. P. oxalicum grows and survives better in drought conditions regardless of temperature and soil type. P. oxalicum grows and survives more in sandy loam soils with low organic matter content, higher pH and lower availability of phosphorus and nitrogen. Survival and growth of P. oxalicum negatively correlates with the availability of water and positively with the organic content. Only survival also correlated positively with pH. Moreover, trials are carried out into commercial orchards soils with different physic-chemical properties and different environmental conditions TO STUDY THE ESTABLISHMENT, SURVIVAL, VERTICAL DISPERSION AND HORIZONTAL SPREAD OF PO212. P. oxalicum 212 can persist and survive at very low levels in soil one year after its release. The size of the PO212 population after its release into the tested natural soils is similar to that of indigenous Penicillium spp. Furthermore, the vertical dispersion and horizontal spread of PO212 is limited in different soil types. The introduction of P. oxalicum in a natural environment not only involves their action on the target organism, the pathogen, but also on other indigenous microorganisms. TO ASSESS THE EFFECT OF P. oxalicum APPLICATION ON SOIL INDIGENOUS FUNGAL COMMUNITIES in two commercial orchards, soil samples are analyzed by Denaturing Gradient Gel Electrophoresis polyacrylamide (DGGE). Samples are taken from soil at two depths (5 and 10 cm) and four dates from the application of P. oxalicum 212 (0, 75, 180 and 365 days). DGGE analysis shows that differences are observed between sampling dates and are independent of the treatment of P. oxalicum applied and the depth. BCA application does not affect the fungal population of the two soil analyzed. Sequence analysis of the DGGE bands confirms previous findings and to identify the presence of BCA on soils. The presence of P. oxalicum in soil is especially related to environmental factors such as humidity. Therefore, we conclude that the 212 of strain Penicillium oxalicum can be considered an optimum BCA, since it is environmentally competitive and effective against a broad spectrum of diseases and does not have any negative effect on soil non-target fungi communities.

Electronic states of trans-polyacetylene, poly(p-phenylene vinylene) and sp-hybridised carbon species in amorphous hydrogenated carbon probed by resonant Raman scattering

Inclusions of sp-hybridised, trans-polyacetylene [trans-(CH)x] and poly(p-phenylene vinylene) (PPV) chains are revealed using resonant Raman scattering (RRS) investigation of amorphous hydrogenated carbon (a-C:H) films in the near IR – UV range. The RRS spectra of trans-(CH)x core Ag modes and the PPV CC-H phenylene mode are found to transform and disperse as the laser excitation energy ћωL is increased from near IR through visible to UV, whereas sp-bonded inclusions only become evident in UV. This is attributed to ћωL probing of trans-(CH)x chain inhomogeneity and the distribution of chains with varying conjugation length; for PPV to the resonant probing of phelynene ring disorder; and for sp segments, to ћωL probing of a local band gap of end-terminated polyynes. The IR spectra analysis confirmed the presence of sp, trans-(CH)x and PPV inclusions. The obtained RRS results for a-C:H denote differentiation between the core Ag trans-(CH)x modes and the PPV phenylene mode. Furthermore, it was found that at various laser excitation energies the changes in Raman spectra features for trans-(CH)x segments included in an amorphous carbon matrix are the same as in bulk trans-polyacetylene. The latter finding can be used to facilitate identification of trans-(CH)x in the spectra of complex carbonaceous materials.