Effects of Thaumatin-like proteins, chitinase and plant secondary metabolites on Botrytis cinerea development

In this work the ability of the necrotrophic plant pathogen Botrytis cinerea to counteract two plants antimicrobial proteins – a chitinase and a grape thaumatin-like protein (TLP) from grape– was characterized. These two proteins extracted from grape berries (cv. IM 6.0.13) were added to the B. cinerea culture. The growth of the fungus was not affected by these proteins which, as determined by RP-HPLC, were partially removed from the medium. It was observed that aspartic and serine protease produced by B. cinerea cleave chitinase only, while the B. cinerea mycelium absorbs passively both proteins. In fact, both chitinase and TLP were released from the fungal cell wall when the mycelium was treated with an alkaline buffer and a laminarinase (β-1,3 glucanase) preparation. In particular, a stronger interaction between chitinase, TLP and a β-1,3-1,6-glucan polymer (scleroglucan) was observed. Therefore, B. cinerea can entrap TLP and chitinase in its cell wall, and particularly by the layer of β-glucan that surrounds fungal hyphae. To investigate if TLP and chitinase can induce the synthesis of new fungal cell wall material, the expression of β glucan synthase, chitin synthase and chitin deacetylases was investigated. The expression of the β glucan synthase genes was not affected by both TLP and chitinase while the expression of genes encoding chitin synthase and chitin deacetylases, responsible for the synthesis of chitin and chitosan, respectively, was lowered following the treatment with the grape proteins. Localization experiments of the cell wall components in the mycelium of B. cinerea showed that chitin is mainly present in the vegetative hyphae, chitosan predominate in infection cushions and β-glucan is evenly distributed. A model describing the mechanisms by which B. cinerea could escape the action of plant TLP and chitinase is proposed.In questo lavoro è stata studiata la capacità del fungo necrotrofo Botrytis cinerea di contrastare l’effetto di due proteine antimicrobiche – una chitinasi e una proteina thaumatin-like (TLP) di vite – presenti in elevata quantità negli acini di uva. Queste proteine estratte dall’uva (Vitis vinifera cv. IM 6.0.13) e somministrate alle colture di B. cinerea non hanno alterato la crescita del fungo neppure ad alte concentrazioni delle due proteine. Analisi mediante RP-HPLC hanno dimostrato che TLP e chitinasi vengono rimosse dal mezzo di coltura. Le attività aspartil- e serin-proteasica prodotte da B. cinerea in presenza di proteine dell’uva sono in grado di degradare la chitinasi ma non la TLP. Il micelio di B. cinerea, invece, si è dimostrato capace di adsorbire passivamente ambedue le proteine di vite. Infatti, dopo trattamento con un buffer alcalino e con β-1,3-glucanasi, il micelio rilascia significative quantità di TLP e chitinasi. In particolare una forte interazione è stata osservata tra le due proteine e il β-1,3-1,6-glucano (scleroglucano) di parete del fungo. Queste evidenze suggeriscono un ruolo protettivo dei β-glucani che rivestono le ife del fungo dall’azione delle proteine della pianta. Per verificare se TLP e chitinasi inducono la sintesi di nuovo materiale di parete, è stata studiata l’espressione della β-glucan sintasi, della chitin sintasi e della chitin deacetilasi di B. cinerea in seguito al trattamento con TLP e chitinasi. Queste analisi hanno evidenziato che il livello di espressione della β-glucan sintasi di B. cinerea non è influenzato dal trattamento con TLP e chitinasi mentre l’espressione dei geni codificanti chitin sintasi e chitin deacetilasi – responsabili, rispettivamente, della sintesi di chitina e chitosano – diminuiscono in seguito al trattamento con le proteine dell’uva. Esperimenti di localizzazione nel micelio di B. cinerea dei polimeri di parete hanno evidenziato che la chitina è presente principalmente nelle ife vegetative, il chitosano prevale nei cuscinetti di infezione e i β-glucani sono presenti ubiquitariamente. I risultati ottenuti consentono di proporre un modello che descrive i meccanismi con cui B. cinerea può sfuggire all’azione della TLP e della chitinasi di pianta

L'insertion des jeunes en France et en Italie : politiques d'activation, stratégies institutionnelles et dynamiques professionnelles.

In Europe the subsidiarisation process of social policies and employment strategy impacts the local networks of actors linked to the development of new active policies. Its consequences are managed daily by professionals working with citizens. They explain to users fractures of a welfare state in transition - from a socialization of collective risks to an individual responsibility of protection- and they give meaning to the institutional boundaries , geographical and social , built between active political devices.The research question of this thesis is to show how the European model of active employment policy structure practices and operations of new premises of welfare systems. To answer it discusses the strategies of actors face the contradictions caused by the rhetorical dimension of the new active welfare and the practical dimension of the crisis of funding. It is therefore addressing the issue of flexibilities of street level bureaucrats to deal with change and make sense of often remote European directives intervention courses and characteristics of target audiences.At the theoretical level, the objective of this research is to propose a conceptual framework to explaining the process of subsidiarisation and activation of the welfare state from its effects. Our working hypothesis leads us to explore a correlation between active employment policies and : 1. the territorial fragmentation of institutions and stakeholder networks ; 2. the specialization of public employment services; 3. the new instruments of contracts ; 4. professionalization and casualization of stakeholders.Using a comparative research in France and Italy, we offer an analysis of professional strategies involved in social integration policies for young people. About youth policy, they show the components of the local systems of welfare (Loncle, 2011) and draw welfare conditions for the new generations. In addition, since the implementation of the European Employment Strategy in 1997 and the European Youth Pact in 2005, they indicate the transition arrangements to adulthood and define the criteria of employability in the market European employment. We prefer the specific method thumbnails (Barter and Renold, 2000; Finch, 1987; Hughes and Huby, 2002) to show the scenes and actors strategies in the activation process. We complet these survey materials with semi-structured interviews and participant observation to examine different levels of actors, institutions and professionals related to the restructuring of the local welfare.En Europe le processus de subsidiarisation des politiques sociales et de l’emploi (Kazepov, 2010) impacte fortement les réseaux locaux d’acteurs liés à la mise en place de nouvelles politiques actives (van Berkel et Borghi, 2007 ; Heidenreich et Rice, 2016). Ses conséquences sont gérées au quotidien par les professionnels intervenant auprès des citoyens. Ils expliquent aux usagers les fractures d’un Etat social en phase de transition – d’une socialisation des risques collectifs à une responsabilité individuelle de la protection (Bresson, Colomb et Gaspar, 2015 ; Andreotti, Mingione et Polizzi, 2012) – et ils donnent du sens aux frontières institutionnelles, géographiques et sociales (Ferrera, 2005), construites entre les dispositifs des politiques actives.La question de recherche de cette thèse vise à montrer en quoi le modèle européen de politiques actives de l’emploi structure les pratiques et les fonctionnements de nouveaux systèmes locaux de welfare. Pour y répondre elle aborde les stratégies d’acteurs face aux antinomies causées par la dimension rhétorique du nouveau welfare actif (Bifulco, 2005) et la dimension pratique de la crise des financements. Il s’agit donc de traiter la question des marges de manœuvre des street level bureaucrates (Lipsky, 1980) pour faire face au changement et donner du sens à des directives européennes souvent éloignées des terrains d’intervention et des caractéristiques des publics cibles.Au niveau théorique, l’objectif de ce travail de recherche est de proposer un cadre conceptuel capable d’expliquer le processus de subsidiarisation et de l’activation de l’Etat social à partir de ses effets. Notre hypothèse de travail nous amène à explorer une corrélation entre les politiques actives de l’emploi et : 1. la fragmentation territoriale des institutions et des réseaux d’acteurs ; 2. la spécialisation des services publics de l’emploi ; 3. les nouveaux instruments de contractualisation ; 4. la professionnalisation et précarisation des intervenants.A l’aide d’une recherche comparative en France et en Italie, nous proposons une analyse des stratégies professionnelles intervenant dans les politiques sociales d’insertion en faveur des jeunes. A propos de politiques de jeunesse, elles nous montrent les composantes des systèmes locaux de welfare (Loncle, 2011) et dessinent les modalités de protection sociale pour les nouvelles générations. En outre, depuis la mise en place de la Stratégie Européenne de l’Emploi en 1997 et le Pacte Européen pour la jeunesse en 2005, elles indiquent les modalités de transition vers l’âge adulte et définissent les critères d’employabilité dans le marché de l’emploi européen. Nous privilégions la méthode spécifique des vignettes (Barter et Renold, 2000 ; Finch, 1987 ; Hughes et Huby, 2002) afin de montrer les coulisses et les stratégies d’acteurs dans le processus d’activation. Nous complétons ces matériaux d’enquête avec des entretiens semi-directifs et des observations participantes visant à interroger différents niveaux d’acteurs, institutionnels et professionnels, liés à la restructuration du welfare local

Youth employment in France and Italy : activation policies, institutional strategies and professionnal dynamics

En Europe le processus de subsidiarisation des politiques sociales et de l’emploi (Kazepov, 2010) impacte fortement les réseaux locaux d’acteurs liés à la mise en place de nouvelles politiques actives (van Berkel et Borghi, 2007 ; Heidenreich et Rice, 2016). Ses conséquences sont gérées au quotidien par les professionnels intervenant auprès des citoyens. Ils expliquent aux usagers les fractures d’un Etat social en phase de transition – d’une socialisation des risques collectifs à une responsabilité individuelle de la protection (Bresson, Colomb et Gaspar, 2015 ; Andreotti, Mingione et Polizzi, 2012) – et ils donnent du sens aux frontières institutionnelles, géographiques et sociales (Ferrera, 2005), construites entre les dispositifs des politiques actives.La question de recherche de cette thèse vise à montrer en quoi le modèle européen de politiques actives de l’emploi structure les pratiques et les fonctionnements de nouveaux systèmes locaux de welfare. Pour y répondre elle aborde les stratégies d’acteurs face aux antinomies causées par la dimension rhétorique du nouveau welfare actif (Bifulco, 2005) et la dimension pratique de la crise des financements. Il s’agit donc de traiter la question des marges de manœuvre des street level bureaucrates (Lipsky, 1980) pour faire face au changement et donner du sens à des directives européennes souvent éloignées des terrains d’intervention et des caractéristiques des publics cibles.Au niveau théorique, l’objectif de ce travail de recherche est de proposer un cadre conceptuel capable d’expliquer le processus de subsidiarisation et de l’activation de l’Etat social à partir de ses effets. Notre hypothèse de travail nous amène à explorer une corrélation entre les politiques actives de l’emploi et : 1. la fragmentation territoriale des institutions et des réseaux d’acteurs ; 2. la spécialisation des services publics de l’emploi ; 3. les nouveaux instruments de contractualisation ; 4. la professionnalisation et précarisation des intervenants.A l’aide d’une recherche comparative en France et en Italie, nous proposons une analyse des stratégies professionnelles intervenant dans les politiques sociales d’insertion en faveur des jeunes. A propos de politiques de jeunesse, elles nous montrent les composantes des systèmes locaux de welfare (Loncle, 2011) et dessinent les modalités de protection sociale pour les nouvelles générations. En outre, depuis la mise en place de la Stratégie Européenne de l’Emploi en 1997 et le Pacte Européen pour la jeunesse en 2005, elles indiquent les modalités de transition vers l’âge adulte et définissent les critères d’employabilité dans le marché de l’emploi européen. Nous privilégions la méthode spécifique des vignettes (Barter et Renold, 2000 ; Finch, 1987 ; Hughes et Huby, 2002) afin de montrer les coulisses et les stratégies d’acteurs dans le processus d’activation. Nous complétons ces matériaux d’enquête avec des entretiens semi-directifs et des observations participantes visant à interroger différents niveaux d’acteurs, institutionnels et professionnels, liés à la restructuration du welfare local.In Europe the subsidiarisation process of social policies and employment strategy impacts the local networks of actors linked to the development of new active policies. Its consequences are managed daily by professionals working with citizens. They explain to users fractures of a welfare state in transition - from a socialization of collective risks to an individual responsibility of protection- and they give meaning to the institutional boundaries , geographical and social , built between active political devices.The research question of this thesis is to show how the European model of active employment policy structure practices and operations of new premises of welfare systems. To answer it discusses the strategies of actors face the contradictions caused by the rhetorical dimension of the new active welfare and the practical dimension of the crisis of funding. It is therefore addressing the issue of flexibilities of street level bureaucrats to deal with change and make sense of often remote European directives intervention courses and characteristics of target audiences.At the theoretical level, the objective of this research is to propose a conceptual framework to explaining the process of subsidiarisation and activation of the welfare state from its effects. Our working hypothesis leads us to explore a correlation between active employment policies and : 1. the territorial fragmentation of institutions and stakeholder networks ; 2. the specialization of public employment services; 3. the new instruments of contracts ; 4. professionalization and casualization of stakeholders.Using a comparative research in France and Italy, we offer an analysis of professional strategies involved in social integration policies for young people. About youth policy, they show the components of the local systems of welfare (Loncle, 2011) and draw welfare conditions for the new generations. In addition, since the implementation of the European Employment Strategy in 1997 and the European Youth Pact in 2005, they indicate the transition arrangements to adulthood and define the criteria of employability in the market European employment. We prefer the specific method thumbnails (Barter and Renold, 2000; Finch, 1987; Hughes and Huby, 2002) to show the scenes and actors strategies in the activation process. We complet these survey materials with semi-structured interviews and participant observation to examine different levels of actors, institutions and professionals related to the restructuring of the local welfare

Fungitoxic activity of eugenol against Botryitis cinerea may be mediated by fungal peroxidase.

Plant essential oils have shown antifungal activity against several plant pathogenic fungi in pre- and post-harvest. This activity is due to their monoterpenic constituents like eugenol. However, eugenol mechanism of action is not completely clear. We studied the effect of eugenol on Botrytis cinerea (strain B05.10). The effective concentration of eugenol in inhibiting mycelium growth and conidia germination was 1-2 mM and the minimum effective dose of eugenol to protect grape leaves from B. cinerea infection was about 10 mM. Experimental data showed that eugenol causes a release in the B. cinerea culture filtrate of potassium ions and of intracellular material, indicating a damage to cell membrane possibly due to an oxidative stress. However, in comparison to untreated control, no accumulation of reactive oxygen species (measured by xylan orange assay) was observed in B. cinerea mycelia treated with eugenol, while the concentration of antioxidant molecules such as glutathione and cysteine decreased. Since B. cinerea produces high level of peroxidase activity and eugenol is a good competitive substrate of cellular antioxidants, the eugenol activity may be ascribed to the release of hydroxyl radical, a product of the reaction of eugenol with hydrogen peroxide. Moreover, the oxidized eugenol showed the same fungitoxic activity of the not oxidized eugenol

Inhibitory activity of essential oils components against the fungal plant pathogen Botrytis cinerea

Botrytis cinerea is the fungus responsible of the grey mould disease of grape and other fruits. Chemical control is necessary when environmental conditions are favorable to the disease. In order to protect plants from disease the European Union by directive 2009/128/EC gives priority to non-chemical methods and encourages the use of natural non-toxic substances. Natural compounds are used as an alternative to chemical fungicides mostly to control post-harvest diseases. The major aim of this work is to compare the fungicidal activity against B. cinerea of 11 natural compounds that are produced in plants through the shikimate/phenylalanine pathway. In a spore germination assay six compounds (eugenol, thymol, carvacrol, isoeugnol, cinnamaldehyde and vanillin) completely inhibited the germination of conidia. These natural products also reduced the viability of the spores and inhibited the growth of B. cinerea mycelium (strain PM10) even if with different efficiency. These compounds are the major constituents of essential oils of many aromatic species. Some chemical characteristics, like the high lipophilic and the low value of polar surface area, are common among these compounds indicating an high affinity for phospholipids compartment and their difficult to overcome the external layer of the cell