Techn. Universiẗat, Diss., 2004--Berlin.

In the literary part of the thesis are information about the possibilities of influencing the quality of grain barely with foliar fertilizers and elicitors. In the practical part we explored the effect off ertilizer of company AGRA GROUP a.s. on quality of barley. The results of small-plot field trials established on plots of the Agrospol agricultural enterprise in 2014 and 2015 in Velká Bystřic we can see the positive effect of applied foliar fertilizers on the yield of the grain of spring barley variety Bojos. Also we can see positive effect on the quality of the grain, specially on the mechanical properties. Preparation NanoFyt Si had the positive effect on the yield, increase the proportion of leading grain and contributed to higher starch content. Foliar fertilizer K-Gel supported the use of fertilizers UREAstabil + AmisaN, which led to higher yields and better grain quality. Preparation nitrophenol increased yield and starch content of the grain.

Momordica charantia é uma espécie de planta pertencente à família Cucurbitaceae. No Brasil, é conhecida pelo nome popular de Melão de São Caetano. É encontrada em grande parte do globo terrestre, sendo que no Brasil é observada nas regiões litorâneas e principalmente nas regiões do interior do país. Escolhemos o estudo com Momordica charantia devido suas propriedades terapêuticas conhecidas serem de grande interesse. Ela vem ganhando cada vez mais reconhecimento através de pesquisas científicas realizadas, visto que, já é utilizada a milhares de anos através da cultura popular. Tais pesquisas têm confirmado e entendido cada vez mais as propriedades medicamentosas desta planta e os mecanismos responsáveis por tais funções nesta espécie, sendo as propriedades antidiabéticas, antivirais e anticancerígenas as de maior destaque. Os experimentos realizados em torno da Momordica charantia, na busca destes compostos foram todos realizados utilizando extratos retirados dos próprios tecidos já diferenciados na planta adulta (sementes, folhas, raízes e frutos). Neste trabalho, foi realizado a pesquisa de metabólitos secundários produzidos pela cultura de células em suspensão de Momordica charantia durante a fase de crescimento desta e após a realização de ensaios de elicitação com Ácido Salicílico (AS). Nosso laboratório vem se dedicando aos estudos da composição da parede celular vegetal e suas alterações, bem como dos mecanismos de defesa empregados pelas células em suspensão em condições de estresse biótico e abiótico. O ácido salicílico é um dos hormônios vegetais envolvido em vários mecanismos de respostas que envolve o estresse vegetal e de desenvolvimento da planta tais como o de garantir resistência a patógenos por exemplo. Quando ocorre algum processo de infecção ou lesivo para a planta, ocorre um aumento na concentração de AS no interior da planta. Esse aumento leva a ativação de certas vias metabólicas que culminam na produção de metabólitos secundários e na degradação, fragmentação ou espessamento da parede celular. Como reposta dos ensaios de elicitação realizados podemos comprovar os efeitos do AS mediados na produção de metabólitos secundários que foram aumentados conforme a concentração de AS. Foi utilizado para os ensaios de elicitação AS nas concentrações de 0,5 , 1,0 e 5 mmol/L. Para o monitoramento da cultura e averiguação da efetividade dos ensaios de elicitação foi utilizado a monitoria através da dosagem de proteínas totais, compostos fenólicos totais e açúcares redutores totais tanto liberados para o meio extracelular quanto produzidos no meio intracelular. Foi determinado as variações dos componentes da parede celular durante a fase de crescimento e após os ensaios de elicitação onde foi comprovado que quanto maior a concentração de AS, maiores são os fragmentos de açúcares fragmentados da parede celular. Foi evidenciado também a presença de triterpenos na cultura celular (em comparação com os padrões isolados da planta), tanto na fase de crescimento e também após a indução com os ensaios de elicitação com AS. / Momordica charantia is a plant from the Cucurbitaceae family. In Brazil, it is known by the popular name of Melão de São Caetano. It is found across the whole world, and in Brazil it can be found in coastal and interior regions of the country. The aim to study Momordica charantia is due to its known therapeutic properties that are already used for thousands of years through popular culture. The studies have confirmed and understood various drug-like properties of this plant and the mechanisms responsible for such functions in this species, such as antidiabetic, antiviral and anticancer properties. The researches with Momordica charantia were all performed using extracts obtained from seeds, leaves, roots and fruit. In this work, the isolation and characterization of metabolites as standards from leaves and stems of Momordica charantia was performed. The research of secondary metabolites produced in cell culture suspension of Momordica charantia without elicitor or after elicitation with salicylic acid (SA) was conducted. Salicylic acid is a plant hormone involved in various mechanisms of responses. When the plant suffers an infection or injury, it is followed by an increase in AS concentration. This increase leads to activation of certain metabolic pathways that culminate in the production of secondary metabolites and degradation, fragmentation or cell wall thickening. AS was utilized for the elicitation experiments at 0.5, 1.0 and 5 mmol/L. For the monitoring of plant cell culture, it was determinate the total protein, phenolic compounds and reducing sugars content, into the extra and intra cellular medium. Where it was verified that AS modulates the production of these molecules in culture. After this, we determined the variation in the oligosaccharides in the extracellular medium obtained in the presence or absence of AS. It was observed the degree of polymerization of oligosaccharides higher when increase AS concentration in the medium. In conclusion, this study can demonstrate the effects of SA on the production of secondary metabolites in plant cell culture, which were increased as the concentration of AS increased. The presence of triterpenes in cell culture was confirmed to compare the molecules obtained from culture with that isolated from plant standards.

Momordica charantia é uma espécie de planta pertencente à família Cucurbitaceae. No Brasil, é conhecida pelo nome popular de Melão de São Caetano. É encontrada em grande parte do globo terrestre, sendo que no Brasil é observada nas regiões litorâneas e principalmente nas regiões do interior do país. Escolhemos o estudo com Momordica charantia devido suas propriedades terapêuticas conhecidas serem de grande interesse. Ela vem ganhando cada vez mais reconhecimento através de pesquisas científicas realizadas, visto que, já é utilizada a milhares de anos através da cultura popular. Tais pesquisas têm confirmado e entendido cada vez mais as propriedades medicamentosas desta planta e os mecanismos responsáveis por tais funções nesta espécie, sendo as propriedades antidiabéticas, antivirais e anticancerígenas as de maior destaque. Os experimentos realizados em torno da Momordica charantia, na busca destes compostos foram todos realizados utilizando extratos retirados dos próprios tecidos já diferenciados na planta adulta (sementes, folhas, raízes e frutos). Neste trabalho, foi realizado a pesquisa de metabólitos secundários produzidos pela cultura de células em suspensão de Momordica charantia durante a fase de crescimento desta e após a realização de ensaios de elicitação com Ácido Salicílico (AS). Nosso laboratório vem se dedicando aos estudos da composição da parede celular vegetal e suas alterações, bem como dos mecanismos de defesa empregados pelas células em suspensão em condições de estresse biótico e abiótico. O ácido salicílico é um dos hormônios vegetais envolvido em vários mecanismos de respostas que envolve o estresse vegetal e de desenvolvimento da planta tais como o de garantir resistência a patógenos por exemplo. Quando ocorre algum processo de infecção ou lesivo para a planta, ocorre um aumento na concentração de AS no interior da planta. Esse aumento leva a ativação de certas vias metabólicas que culminam na produção de metabólitos secundários e na degradação, fragmentação ou espessamento da parede celular. Como reposta dos ensaios de elicitação realizados podemos comprovar os efeitos do AS mediados na produção de metabólitos secundários que foram aumentados conforme a concentração de AS. Foi utilizado para os ensaios de elicitação AS nas concentrações de 0,5 , 1,0 e 5 mmol/L. Para o monitoramento da cultura e averiguação da efetividade dos ensaios de elicitação foi utilizado a monitoria através da dosagem de proteínas totais, compostos fenólicos totais e açúcares redutores totais tanto liberados para o meio extracelular quanto produzidos no meio intracelular. Foi determinado as variações dos componentes da parede celular durante a fase de crescimento e após os ensaios de elicitação onde foi comprovado que quanto maior a concentração de AS, maiores são os fragmentos de açúcares fragmentados da parede celular. Foi evidenciado também a presença de triterpenos na cultura celular (em comparação com os padrões isolados da planta), tanto na fase de crescimento e também após a indução com os ensaios de elicitação com AS. / Momordica charantia is a plant from the Cucurbitaceae family. In Brazil, it is known by the popular name of Melão de São Caetano. It is found across the whole world, and in Brazil it can be found in coastal and interior regions of the country. The aim to study Momordica charantia is due to its known therapeutic properties that are already used for thousands of years through popular culture. The studies have confirmed and understood various drug-like properties of this plant and the mechanisms responsible for such functions in this species, such as antidiabetic, antiviral and anticancer properties. The researches with Momordica charantia were all performed using extracts obtained from seeds, leaves, roots and fruit. In this work, the isolation and characterization of metabolites as standards from leaves and stems of Momordica charantia was performed. The research of secondary metabolites produced in cell culture suspension of Momordica charantia without elicitor or after elicitation with salicylic acid (SA) was conducted. Salicylic acid is a plant hormone involved in various mechanisms of responses. When the plant suffers an infection or injury, it is followed by an increase in AS concentration. This increase leads to activation of certain metabolic pathways that culminate in the production of secondary metabolites and degradation, fragmentation or cell wall thickening. AS was utilized for the elicitation experiments at 0.5, 1.0 and 5 mmol/L. For the monitoring of plant cell culture, it was determinate the total protein, phenolic compounds and reducing sugars content, into the extra and intra cellular medium. Where it was verified that AS modulates the production of these molecules in culture. After this, we determined the variation in the oligosaccharides in the extracellular medium obtained in the presence or absence of AS. It was observed the degree of polymerization of oligosaccharides higher when increase AS concentration in the medium. In conclusion, this study can demonstrate the effects of SA on the production of secondary metabolites in plant cell culture, which were increased as the concentration of AS increased. The presence of triterpenes in cell culture was confirmed to compare the molecules obtained from culture with that isolated from plant standards.

De nos jours, les agents pathogènes des plantes, engendrent encore des pertes agricoles importantes. A terme, la solution la plus adaptée semble être la création de variétés de plantes cultivées présentant une résistance génétique à large spectre et durable. Créer de telles plantes nécessite la compréhension des mécanismes de résistance mis en jeu dans des pathosystèmes modèles, tels que celui impliquant le gène de résistance Rx et le virus X de la pomme de terre. Dans ce système, la protéine Rx intervient dans la perception d’un facteur d'avirulence issu du virus: sa protéine de capside (Cp). Cependant, les mécanismes moléculaires déclenchant la résistance chez la plante exprimant Rx demeurent obscurs. Cette étude s’est focalisée sur l’étape de reconnaissance de l’éliciteur viral par Rx. Une première approche a permis la mise en évidence d’un éliciteur minimal de 90 acides amine. L’étude d’un fragment légèrement plus grand par des méthodes de biochimie structurale, tend à exclure un modèle selon lequel, la différence de structuration tertiaire entre Cp élicitrice et Cp non élicitrice déterminerait la reconnaissance par Rx. Une seconde approche, de biologie moléculaire, a mis en évidence deux protéines hôte interagissant avec ce petit fragment éliciteur. L'étude s’est focalisée sur un gène codant un facteur de transcription, nbERF5, et a révélé que cette protéine interagit aussi bien avec les Cp de souches de Potexvirus avirulentes que virulentes dans la résistance liée à Rx, mais également avec la protéine de résistance Rx et l’intéracteur direct: RanGAP2. La poursuite de la caractérisation de cet ERF, permettra de déterminer son importance dans la résistance liée à Rx. / Nowadays, phytopathogenic agents are still causing significant agricultural losses. The most suitable option appears to be the creation of crop species carrying a genetic durable and broad spectrum resistance. In order to create such varieties, we need to understand the mechanisms underlying resistance, involved in model Pathosystems, Such as the one composed of the resistance gene Rx and the potato virus X. In that system, the host gene encodes a protein assimilated to a receptor implicated in the perception of an avirulence factor produced by the virus: its capside protein (Cp). Nevertheless, the molecular mechanisms triggering the resistance remain largely unknown. This study has been focused on the elicitor recognition mediated by Rx. A first approach led to the identification of a minimal elicitor containing 90 amino acids has. The structural characterization of a slightly larger protein fragment using biochemical methods suggested that the difference in the tertiary structuration of both elicitor and non-elicitor Cp would not be the determinant of Rx mediated recognition. Second, a molecular approach led to the discovery of two host proteins interacting with the small elicitor fragment. The work was focused on a transcription factor, nbERF5 and showed that this protein interacts similarly with elicitor or non-elicitor Cps of Rx mediated resistance. Interestingly, this gene product is able to directly interact with the Rx protein, but also with the direct interactor of Rx: RanGAP2, protein required for the Rx mediated resistance efficiency. Further characterization of this ethylen response factor will help us to understand its role in Rx mediated resistance.

The soil-borne filamentous fungus Trichoderma virens is a biocontrol agent with a well known ability to produce antibiotics, parasitize pathogenic fungi and induce systemic resistance in plants. Here we report the identification, purification and characterization of an elicitor secreted by T. virens; a small protein designated Sm1 (small protein 1). Confrontation and disk assays demonstrated that Sm1 lacks toxic activity against plants and microbes. Native, purified Sm1 triggers production of reactive oxygen species in rice (Oryza sativa) and cotton (Gossypium hirsutum), and induces the expression of defense related genes both locally and systemically in cotton. Gene expression analysis revealed that SM1 is expressed throughout fungal development and is transcriptionally regulated by nutrient conditions and the presence of a host plant. When T. virens was co-cultured with cotton in an axenic hydroponic system, SM1 expression and secretion of the protein was significantly higher than when the fungus was grown alone. These results indicate that Sm1 is involved in plant-Trichoderma recognition and the induction of resistance by activation of plant defense mechanisms. Following the cloning of SM1, strains disrupted in or over-expressing SM1 were generated. Targeted gene disruption revealed that SM1 was not involved in fungal development. Expression of defense related genes in cotton and maize (Zea mays) was induced locally and systemically following colonization by T. virens in the hydroponic system. Low levels of expression of cotton or maize defense genes were found when seedlings were grown with a T. virens strain disrupted in SM1, ssupporting the Sm1-elicitor hypothesis. Additionally, unique proteins in T.virens-cotton/maize interaction were identified. Thus, the induction of defense responses in two agriculturally important crops appears to be microbially mediated. Functional analysis of a cell wall degrading enzyme, beta-1,6-glucananse (Tv-bgn3) from T. virens, demonstrated involvement of this enzyme indirectly in mycoparasitic activity of T. virens. Protein extracts from the strain disrupted in TV-BGN3 displayed reduced capability to inhibit growth of Pythium ultimum as compared to the wild-type. Additionally, protein extracts from the strains co-expressed with TV-BGN2 (beta-1,3-glucananse) from T. virens showed a significantly increased capability to inhibit growth of P. ultimum and Rhizoctonia solani hyphae.

The soil-borne filamentous fungus Trichoderma virens is a biocontrol agent with a well known ability to produce antibiotics, parasitize pathogenic fungi and induce systemic resistance in plants. Here we report the identification, purification and characterization of an elicitor secreted by T. virens; a small protein designated Sm1 (small protein 1). Confrontation and disk assays demonstrated that Sm1 lacks toxic activity against plants and microbes. Native, purified Sm1 triggers production of reactive oxygen species in rice (Oryza sativa) and cotton (Gossypium hirsutum), and induces the expression of defense related genes both locally and systemically in cotton. Gene expression analysis revealed that SM1 is expressed throughout fungal development and is transcriptionally regulated by nutrient conditions and the presence of a host plant. When T. virens was co-cultured with cotton in an axenic hydroponic system, SM1 expression and secretion of the protein was significantly higher than when the fungus was grown alone. These results indicate that Sm1 is involved in plant-Trichoderma recognition and the induction of resistance by activation of plant defense mechanisms. Following the cloning of SM1, strains disrupted in or over-expressing SM1 were generated. Targeted gene disruption revealed that SM1 was not involved in fungal development. Expression of defense related genes in cotton and maize (Zea mays) was induced locally and systemically following colonization by T. virens in the hydroponic system. Low levels of expression of cotton or maize defense genes were found when seedlings were grown with a T. virens strain disrupted in SM1, ssupporting the Sm1-elicitor hypothesis. Additionally, unique proteins in T.virens-cotton/maize interaction were identified. Thus, the induction of defense responses in two agriculturally important crops appears to be microbially mediated. Functional analysis of a cell wall degrading enzyme, beta-1,6-glucananse (Tv-bgn3) from T. virens, demonstrated involvement of this enzyme indirectly in mycoparasitic activity of T. virens. Protein extracts from the strain disrupted in TV-BGN3 displayed reduced capability to inhibit growth of Pythium ultimum as compared to the wild-type. Additionally, protein extracts from the strains co-expressed with TV-BGN2 (beta-1,3-glucananse) from T. virens showed a significantly increased capability to inhibit growth of P. ultimum and Rhizoctonia solani hyphae.

Univ., Diss.--München, 1998.

Nos travaux de recherches de thèse portent sur une étude comparative entre le déclanchement des mécanismes de défense in planta de la plante de melon après élicitation biotique, et un traitement avec un produit ayant des propriétés de Stimulateur des Défenses Naturelles (SDN). Le pathosystème étudié est Cucumis melo - Fusarium Oxysporum melonis. L'éliciteur abiotique SDN utilisé codé FEN560 est un extrait de graines de fenugrec (Trigonella foenum-graecum). Les principaux travaux sont : 1) L'analyse des activités enzymatiques relatives au stress et caractérisation d'enzymes PR. 2) L'étude des métabolites du métabolisme secondaire, notamment la production l'accumulation des composés organiques volatiles (COV). 3) Une étude de l'expression de gènes candidats impliqués dans la production de ces PR-protéines et/ou dans les voie de signalisation est aussi entreprise ; Les principaux résultats se traduisent par (i) une précocité et intensité de l'induction des activités enzymatiques étudiées pour les deux variétés et pour les organes d'un même plant (ii) Le priming est généralement observé au niveau du site de la seconde élicitation (infection FOM) avec une induction remarquable des PR-protéines. (iii) L'élicitation est Systémique, car l'induction se transmet mais à moindre intensité sur les intervalles de temps étudiés. (iv) L'augmentation de l'émission des COV coïncide avec l'induction de la LOX. Il est aussi noté l'émission de nouvelles molécules connus par leurs propriétés antibiotique ou répulsive des insectes herbivores ou attractive des prédateurs d'insectes herbivores. Les résultats sur les expressions des gènes candidats sont corrélés avec les activités enzymatiques et la production des composés organiques. Il en est ressorti que l'induction de la résistance par le traitement par pulvérisation de FEN560 est similaire au phénomène de « primig » dans la résistance systémique induite. / This work is a comparative study between defense mechanisms in melon plant induced by biotic elicitor and treatment by plant extract having simulative proprieties of plant defense mechanisms. The studied pathosystème is Cucumis melo - Fusarium oxysporum fsp melonis. The used plant extract elicitor is named FEN560, it is from fenugreek seeds (Trigonella foenum-graecum). The main works were: 1) Analysis of the enzymatic activities relating to the stress and characterization of PR-proteins sch as peroxidases and chitinases. The lipoxygenase (LOX), the key enzyme of oxylipins biosynthesis and phenylalanine ammonia-lyase, the key enzyme of the phenylpropanoids pathway, were also studyed. 2) The study of the metabolites of the secondary metabolism, in particular accumulation of volatile organic compounds (VOC). 3) A study of expression candidate genes implied in the production of Pr-proteins and the induced resistance pathways is also undertaken. The main results: (i) precocity and intensity of enzymatic activities induction for the two varieties after inoculation of pretreated plants (ii) the priming is generally observed in the site of the second elicitation with a remarkable induction of PR-proteins. (iii) The elicitation induced by FEN560 is systemic; because induction is transmitted between roots and shoots (iv) The modification of VOC emissions coincides with the induction of the LOX activity. It is also noticed the emission of new molecules known for their antibiotic properties or repulsive of the insects herbivorous or attractive of predatory of herbivorous insects. The results on the genes candidates' expression are in general correlated with the enzymatic activities and the production of the volatile organic compounds through LOX activity. In brief, the induction of resistance by the treatment by FEN560 is similar to the phenomenon of "primig" in induced systemic resistance.

Introducción. En el mar Mediterráneo la mayor cantidad de restos vegetales acumulados en la arena pertenecen a Posidonia oceánica (P.oceanica), estos se depositan en los arribazones. Existen diferentes usos de estos arribazones: sellado de vertederos, construcción, farmacia, industria y medio ambiente, aunque hasta la fecha, no hay evidencias de que se estén empleando en el sector de la agricultura, es por ello que el objetivo general de esta tesis fue estudiar la viabilidad del uso de los arribazones de P.oceanica en la agricultura. Experiencia I: empleo de arribazones de Posidonia oceanica como enmienda orgánica. Para dicha experiencia se realizaron 3 ensayos: (1) Evaluación de las características físico-químicas de P.oceanica como enmienda orgánica; (2) Evaluación de la composición de lixiviados procedentes de suelos agrícolas enmendados con P.oceanica y (3) Evaluación del efecto de los lixiviados obtenidos de suelos enmendados con P.oceanica en la germinación de Lycopersicum esculentum Mill. Según la caracterización físico-química de la P. oceanica, esta puede ser empleada como enmienda orgánica de acuerdo con la legislación. La capacidad de retención de agua de los suelos enmendados con P. oceanica aumentó entre un 200 y un 300%, en función del suelo empleado. El suelo ácido, mostró mayores ventajas tras ser enmendado con P.oceanica, ya que elevó el pH del medio hasta los valores de máxima disponibilidad de los nutrientes del suelo y enriqueció la disolución del suelo en Ca, Mg, y K. Para los lixiviados procedentes de los distintos suelos enmendados con P. oceanica tuvo un efecto bioestimulante en la germinación y el crecimiento. Experiencia II: empleo de arribazones de Posidonia oceanica como sustrato de cultivo. Para llevar a cabo esta experiencia se realizaron 2 ensayos: (1) Evaluación de las características físico-químicas de P.oceanica como sustrato de cultivo; (2) Desarrollo de plantas de tomate empleando P. oceanica como sustrato de cultivo. La caracterización físico-química de la P. oceanica, resultó cumplir con la actual legislación como sustrato de cultivo no inerte. Las plantas de tomate cultivadas con P. oceanica cribada (PSL) y cribada y lavada (PL) mostraron síntomas de estrés. En las PSL, pudo ser generado por la elevada salinidad del medio, mientras que para las PL, fue consecuencia de una elevada liberación de metabolitos secundarios. Las plantas PSL mostraron una menor productividad que las desarrolladas en fibra de coco, mientras que en las PL fue similar que para el sustrato control. Como las plantas cultivadas en P.oceanica estaban sometidas a estrés, los frutos presentaron mayores contenidos en compuestos fenólicos y licopeno, aumentando el valor nutricional respecto a los cultivados en fibra de coco. Experiencia III: efecto de la aplicación de extractos acuosos de Posidonia oceanica sobre el desarrollo vegetal. Para dicha experiencia se realizaron 3 ensayos: (1) Obtención y caracterización de extractos vegetales de P. oceanica; (2) Efecto de la aplicación de extractos acuosos de P. oceanica sobre la germinación de semillas de tomate Seny 1 y (3) Efecto de la aplicación foliar de extractos acuosos de P. oceanica a plantas de tomate sometidas a estrés salino. De los dos extractos de P. oceanica, el diluido, estimuló la germinación de las semillas. La aplicación de dicho extracto redujo el estrés oxidativo producido por salinidad, mejorando la integridad de las membranas plasmáticas de las células radiculares, y reduciendo el contenido en prolina, MDA y H2O2 foliar. Estos efectos beneficiosos frente al estrés salino confirmarian la actividad elicitora de los extractos de P.oceanica, debido a la presencia de Si en su composición y compuestos fenólicos.