Philosophiae Doctor - PhD / We hypothesize that South African medicinal plants contain compounds that can act in synergism with synthetic antifungal compounds. Four fungicides - Sporekill™, Rovral™, Terminator™ and Teldor™ at doses 0.1, 0.2, 0.4 and 0.8 mL L-1 and plant species Galenia africana, Elytropappus rhinocerotis and Tulbaghia violacea were tested aloneand in different combinations for their potency (efficacy) on radial growth inhibition of Botrytis cinerea strains on potato dextrose plates. Four doses of plant extract for each of the respective plant species were used. A total of 48 combinations were tested for each strain. Mixtures of plant extracts were far more effective in controlling strains compared to the individual components alone, representing significant levels of in vitro synergistic interactions. Combinations of these components represent an attractive future prospect for the development of new management strategies for controlling B. cinerea. Since the in vitro tests of these mixtures showed inhibitory activity, the mixtures were tested for activity in assays on Granny Smith apples. In vitro tests can be used to screen mixtures to obtain information on their inhibitory activity on a pathogen, however, the environmental conditions of the fruit and the ability of the pathogen to grow into the fruit cannot be simulated in vivo. A series of two-fold doses of medicinal plant extracts were combined with fungicides to conduct decay inhibition studies. The incidence of gray mold was significantly reduced by mixtures of plant extracts and fungicides. Under conditions similar to those in commercial storage, a drench treatment with G. africana and Rovral™ significantly (p=0.05) inhibit gray mold on the apples and was more effective than the plant extract and fungicide alone. The treatments exerted synergistic effects and were markedly better than the components applied alone. The wound colonization assay was used for optimal decay control. In a drench, much higher volumes of the treatments are used to ensure that the components of the suspension are deposited evenly over the entire fruit surface. Drenching of fruit to apply other chemicals is an established practise in the pome (fleshy) fruit industry, and simplifies the commercial application of the mixtures, as no additional infrastructure at commercial packing houses will be required. This approach not only makes it possible to reduce fungicide concentrations while maintaining adequate decay control, but also ensures a reduction of the chemical residue on the fruit. / South Africa

Philosophiae Doctor - PhD / We hypothesize that South African medicinal plants contain compounds that can act in synergism with synthetic antifungal compounds. Four fungicides - Sporekill™, Rovral™, Terminator™ and Teldor™ at doses 0.1, 0.2, 0.4 and 0.8 mL L-1 and plant species Galenia africana, Elytropappus rhinocerotis and Tulbaghia violacea were tested alone and in different combinations for their potency (efficacy) on radial growth inhibition of Botrytis cinerea strains on potato dextrose plates. Four doses of plant extract for each of the respective plant species were used. A total of 48 combinations were tested for each strain. Mixtures of plant extracts were far more effective in controlling strains compared to the individual components alone, representing significant levels of in vitro synergistic interactions. Combinations of these components represent an attractive future prospect for the development of new management strategies for controlling B. cinerea. Since the in vitro tests of these mixtures showed inhibitory activity, the mixtures were tested for activity in assays on Granny Smith apples. In vitro tests can be used to screen mixtures to obtain information on their inhibitory activity on a pathogen, however, the environmental conditions of the fruit and the ability of the pathogen to grow into the fruit cannot be simulated in vivo. A series of two-fold doses of medicinal plant extracts were combined with fungicides to conduct decay inhibition studies. The incidence of gray mold was significantly reduced by mixtures of plant extracts and fungicides. Under conditions similar to those in commercial storage, a drench treatment with G. Africana and Rovral™ significantly (p=0.05) inhibit gray mold on the apples and was more effective than the plant extract and fungicide alone. The treatments exerted synergistic effects and were markedly better than the components applied alone. The wound colonization assay was used for optimal decay control. In a drench, much higher volumes of the treatments are used to ensure that the components of the suspension are deposited evenly over the entire fruit surface

The circadian clock regulates a wide range of biological processes, allowing plants to be prepared for predictable daily diurnal changes in environmental cues such as light and temperature. Recent studies have suggested that the circadian clock may also control plant immunity. The exact nature of the interaction between the circadian clock and plant pathogens remains unknown. Our focus in this study is on the elucidation of the role of the biological clock in plant immunity against the necrotrophic pathogen to Botrytis cinerea. In order to do this we tested the level of susceptibility to B. cinerea in Arabidopsis thaliana wild type and transgenic plants: toc1, cca1/lhy, cca1/toc1, lhy/toc1, cca1/lhy/toc1, GLK1 OE, GLK2 OE, glk1, glk2, and glk1/glk2. We demonstrated that the time of infection plays a role in susceptibility to B. cinerea. Specifically, we found that plants are more susceptible to infection in the subjective morning. We also found that genetic mutations in core clock components or in GLK genes leads to changes in susceptibility to B. cinerea. Our data suggests that clock genes are not solely responsible for plant immune responses to B. cinerea but rather the ways in which the biological clock system regulates outcome pathways. Furthermore, when we entrain the biological clock by changing the photoperiod (day length) in normal earth conditions LD 24h and SD 24h, we observed that short day plants had higher susceptibility to B. cinerea than long day plants. In addition, when we entrain the biological clock in different photoperiods, the LD 30h photoperiod plants displayed similar responses as those in the SD 24h photoperiod. The data indicates that day length is not responsible for the control of plant immunity; it is the ability of light to entrain the biological clock that is important. Together, the data strongly support the conclusion that the circadian clock plays a role in plant defense regulation.

Orientador: Marta Cristina Teixeira Duarte / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-27T13:08:18Z (GMT). No. of bitstreams: 1 Almeida_GustavoSteffende_M.pdf: 767493 bytes, checksum: c194394bcc80ba1852de47976dba16ad (MD5) Previous issue date: 2015 / Resumo: A cultura do morango é de grande importância no contexto agrícola mundial, com uma produção anual que supera 4,5 milhões de toneladas. Porém, as características desta fruta a tornam altamente susceptível a fatores físicos e biológicos de deterioração, tornando-a altamente perecível e acarretando altos níveis de perdas no campo e na pós-colheita. Dentre os fatores biológicos tem destaque a ação de fungos fitopatogênicos, dentre os quais o 'Botrytis cinerea' e o' Colletotrichum gloeosporioides' tem papel principal. Dentre as alternativas naturais para controle destes fitopatógenos, os óleos essenciais de plantas medicinais e aromáticas vêm sendo investigados. Neste contexto, no presente trabalho a atividade antifúngica de óleos essenciais de nove espécies de 'Citrus' e de 'Cymbopogon citratus' foi avaliada in vitro contra 'B. cinerea' e' C. gloeosporioides'. Os óleos de 'Citrus sinensis' e 'C. citratus' foram os mais ativos in vitro (MIC ¿ 0,25 e 0,06 mg.mL-1, respectivamente) e, portanto, selecionados para os ensaios com morangos in natura, visando manter sua qualidade pós-colheita. Os ensaios foram conduzidos sob duas condições ambientais, ou seja, sob condições ambiente de armazenamento (25 ºC) e em armazenamento refrigerado (10 ºC). Aplicados na concentração 1,0 mg.mL-1 , o óleo essencial de 'C. sinensis' mostrou-se mais eficaz sob condições ambiente de armazenamento, e a mistura dos dois óleos em partes iguais em armazenamento refrigerado. Em ambas as condições, melhores resultados foram obtidos através da aplicação por contato direto. Os óleos essenciais foram ainda incorporados a um filme de revestimento composto por matriz de metil-celulose e testados in vitro quanto ao seu potencial de inibição de 'B. cinerea'. Nestas condições, o óleo essencial de 'C. sinensis' mostrou-se o mais eficiente. Através dos resultados foi possível concluir que os óleos essenciais são uma opção viável para o controle de fitopatógenos em morango e para manutenção de sua qualidade pós-colheita / Abstract: The strawberry crop has great importance in agricultural world wide context, with an annual production exceeding 4.5 million tons. However, the characteristics of this fruit make it highly susceptible to physical and biological damage, making it easily perishable and causing losses in field and postharvest. Among the biological factors, the action of phytopathogenic fungi such as 'Botrytis cinerea' and "Colletotrichum gloeosporioides" have a main role. Natural alternatives have been proposed in order to replace synthetic products currently used in control of these plant pathogens, of which the essential oils from medicinal and aromatic plants have demonstrated good potential. In this context, the present study evaluated the in vitro antifungal activity of the essential oils from nine 'Citrus' species and from' Cymbopogon citratus' against 'B. cinerea' and 'C. gloeosporioides'. 'Citrus sinensi's and 'C. citratus' essential oils showed highest activities (MIC values 0.25 mg.mL-1 and 0.06 mg.mL-1, respectively) and were selected for the assays with in natura strawberries aiming at maintaining its postharvest quality. The tests were conducted under two simulated environmental conditions, ie, environment conditions of storage (25 ºC) and refrigerated storage (10 ºC). The crude essential oil of 'C. sinensis' (1.0 mg.mL-1) was the more effective in environmental conditions of storage, and the mixture of the two oils (1:1 ¿ 1.0 mg.mL-1) in refrigerated storage. In both conditions, best results were obtained when the oils were applied by direct contact. The essential oils were also incorporated into a coating film consisting of methyl cellulose matrix and tested to their 'in vitro' inhibitory potential against 'B. cinerea'. Accordingly, the essential oil of 'C. sinensis' proved to be the most effective as antifungal. The results allowed conclude that essential oils are a viable option for controlling phytopathogenic fungi in strawberry and for continuing postharvest quality / Mestrado / Ciência de Alimentos / Mestre em Ciência de Alimentos

Ingeniera Civil en Biotecnología / Chile: Potencia Alimentaria y Forestal es el nuevo slogan adoptado por el país y simboliza el buen escenario del sector silvoagropecuario. Sin embargo, la planta y los frutos de la vid son atacados por el hongo patógeno Botrytis cinerea, produciendo grandes pérdidas productivas y económicas. Nuevas oportunidades de negocio se abren con el cultivo de flores de exportación. Para cuidar que las flores permanezcan en óptimas condiciones se debe combatir el principal patógeno que enferma a estas plantas, la bacteria Erwinia carotovora. Recientemente se ha demostrado que extractos de plantas, algas y esponjas marinas que contienen geranilfenoles y geranilhidroquinonas, poseen propiedades antifúngicas y antibacterianas. Debido a esto y los bajos rendimientos con que se obtienen desde la fuente natural es que se ha decidido sintetizar 6 de estos compuestos en el Laboratorio de Síntesis Orgánica de la UTFSM. Se estudió el efecto de los compuestos H1, M1, I1, I5, L1 y L2 sobre el crecimiento de Botrytis cinerea y Erwinia carotovora. Además, se planteó aumentar la solubilidad de los compuestos y su efectividad contra los patógenos, mediante un sistema de encapsulamiento. Del estudio de actividad antifúngica se obtuvo resultados prometedores para los compuestos, en particular el compuesto H1, que incluso logró porcentajes de inhibición mayores que los del producto comercial Captan, utilizando las mismas concentraciones. H1 a 250 ppm provocó una inhibición del crecimiento de B. cinerea del 86%. También exhibieron altos porcentajes de inhibición del crecimiento del hongo, H1 a 150 ppm con 82% e I1 a 250 ppm con 81%. Al probar la actividad antibacteriana de los compuestos también se obtuvo un buen candidato de estudio, L1. Aunque demostró tener poder bactericida contra Erwinia carotovora a 100 ppm y contra Bacillus subtilis a 50 ppm, esta actividad antibacteriana no fue estable en el tiempo. Sólo fue estable contra Bacillus en el caso en que la infección se realiza al mismo tiempo que la adición del compuesto. El sistema de encapsulamiento fue efectivo en la solubilización de los compuestos pero inefectivo en su uso en pruebas biológicas. Los altos porcentajes de inhibición del crecimiento de Botrytis obtenidos por los compuestos sin encapsular se vieron disminuidos al utilizar los compuestos en su forma encapsulada, llegando a bajar, en el peor de los casos, desde 81% a 26% para I1 a 250 ppm. Al utilizarse el compuesto L1 encapsulado en los ensayos de actividad antibacteriana, en todos los casos, se revirtió el poder bactericida del compuesto, provocando gran proliferación de ambos patógenos, E. carotovora y B. subtilis. Se postula que estos compuestos son muy estables dentro de la micela de encapsulamiento y su liberación es muy lenta como para ser registrada durante la duración de los experimentos. Entre las recomendaciones realizadas para estudios futuros, se hace énfasis en la necesidad de un estudio de cinética de liberación. Se propone el compuesto H1 como componente principal de un potencial producto comercial para combatir el hongo Botrytis cinerea. Para el caso de la actividad antibacteriana, se debe seguir investigando para aumentar la estabilidad del compuesto L1.

Doctor Educationis / The fungus Botrytis cinerea is an opportunistic pathogen on a wide variety of crops, causing a disease known as grey mould through infections via wounds or dead plant parts. Synthetic fungicides for controlling this disease are fast becoming ineffective due to the development of resistance. This, coupled with consumers' world wide becoming increasingly conscious of potential environmental and health problems associated with the build-up of toxic chemicals, (particularly in food products), have resulted in pressure to reduce the use of chemical pesticide volumes as well as its residues. An emerging alternative to random chemical synthesis is the study and exploitation of naturally occurring products with fungicidal properties. One group of compounds known as strobilurins produced by Strobilurus species, woodland basidiomycete fungi, is a good example of this phenomenon. Plants produce an enormous array of secondary metabolites, and it is commonly reasoned that a significant part of this chemical diversity serves to protect plants against plant pathogens. A problem with plant-produced compounds as potential fungicides is that in the natural state, they are generally only weakly active compared to synthetic fungicides. There have been reports on the uses of mixtures of synthetic fungicides for the control of plant pathogenic fungi. When utilized in two-way mixtures, such fungicides may maintain or enhance the level of control of a pathogen at reduced rates for both components utilized in combinations, or alone at normal rates. These studies provide an important precedent for the idea of synergism. For this study, we hypothesize that the addition of plant extracts may enhance the antifungal efficacy of the synthetic strobilurin fungicide, kresoxim-rnethyl against B. cinerea. We selected South African medicinal plant species such as Artemesia afra, Elyptropappus rhinocerotis, Galenia africana, Hypoxis hemerocallidea, Siphonochilus aetheopicus, Sutherlundia frutescence, Tulbaghia violacea and Tulbaghia alliaceae for this study. For the in vitro study, indigenous medicinal plant extracts were prepared at twofold dilution concentrations and combined with kresoxim-rnethyl at concentrations of 0.25 and 0.5% (w/v). The B. cinerea mycelial plug assays showed potent antifungal inhibitory effects with the plant extract and kresoxim-rnethyl mixtures. Further analyses of the mixtures indicate synergistic effects between the fungicide and plant extracts. I surmise that these in vitro effects are also achievable in vivo. Combinations of these agents represent an attractive avenue for the development of new management strategies for controlling B. cinerea in the future. A second study was conducted to analyse the final dose rates for synergistic reactions for combinations of kresoxim-methyl and medicinal plant extracts against B. cinerea in vivo. A series of two-fold concentrations of medicinal plant extracts were combined with kresoxim-methyl to conduct decay inhibition studies on Granny Smith apples. Synergistic effects were observed for many of the kresoxim-methyl and plant extract combinations. I, therefore, came to the conclusion that indigenous South African plant species produce modulators that potentiate the activity of fungicides. Whether these synergistic effects are due to the inhibition of fungal multi-drug resistant pumps require further studies at the molecular level. However, these inhibitory effects are likely to be advantageous for developing fungicide formulations and application strategies with low toxicity effects on the environment. This approach not only makes it possible to reduce fungicide concentrations while maintaining adequate decay control, but also ensures a reduction of the chemical residue on the fruit.

The necrotrophic fungus Botrytis cinerea (B. cinerea) is one of the most aggressive and widespread pathogenic fungi, causing the gray mold disease in over 200 different plants species and severely impacting the quality of harvested fruits. Due to B. cinerea resistance to common synthetic fungicides and their ecological impact, there is a large demand for novel control approaches, such as bio-fungicides. The family of carotenoid-cleavage products, i.e. apocarotenoids, includes important compounds, such as hormones, signaling molecules and growth regulators. Previous studies indicated that the apocarotenoid B-ionone inhibits the growth of some fungal species. In this thesis, we unravel a novel role for B-ionone as a plant regulatory metabolite increasing the resistance against B. cinerea in several plant models. We achieved this by combining phenotypic, transcriptomic, and metabolomic analysis. We show that pretreatment of Arabidopsis plants with B-ionone significantly alleviated the symptoms of B. cinerea infection, modulated hormone homeostasis and affected the interactions between jasmonic acid (JA)/ethylene (ET) and abscisic acid (ABA) hormone signaling pathways β-ionone treatment stimulated JA/ET signaling pathways and repressed the synthesis of ABA upon B. cinerea infection, which reduced the susceptibility of Arabidopsis plants to B. cinerea. To get an overview on the effect of β-ionone on plants at transcript level, we performed an RNA-seq experiment that supported our hypothesis that B-ionone primes and enhances the Arabidopsis immune response to B. cinerea infection and raveled the effect of this volatile on the expression of several transcription factors involved in Arabidopsis immune response to B. cinerea and on transcripts related to cell wall biosynthesis, Map Kinase 3 signaling and hypoxia tolerance. Further experiments performed with transgenic tobacco and tomato plants confirmed the positive role of β-ionone in reducing the symptoms of B. cinerea in green tissues as well as in tomato fruits. The discovery of β-ionone as a new apocarotenoid signaling molecule that regulates plant hormone homeostasis opens up new possibility to control B. cinerea infection and to establish this natural volatile as an environmentally friendly and safe bio-fungicide.

No description available.

Tesis por compendio / [EN] The biocontrol agent (BCA) Candida sake CPA-1, has proven to be effective against the pathogenic fungus Botrytis cinerea, causing agent of grey mold in many fruits. The aim of this Thesis was to develop biocontrol products (BCP), based on this BCA and coating forming agents (CFAs) with good stability and efficacy against fungus infection. Several formulations of CFAs, based on biopolymers (hydroxypropylmethylcellulose (HPMC), corn starch (S), sodium caseinate (NaCas) and pea protein (PP)), combined with surfactants (oleic acid (OA), Span 80 (S80) and Tween 85 (T85)), were obtained and analyzed as to their ability to improve the adherence, survival and efficacy of C. sake on grapes. The functionality of these formulations as coatings was also analyzed with and without yeast cells. Likewise, dry formulations based on low cost CFAs (starch derivatives) and C. sake were obtained by means of fluidized-bed drying and their physical and microbiological stability were studied as a function of product moisture content. The application of C. sake in combination with CFAs permitted an improvement in the initial adherence of the yeast on the surface of grapes and also higher survival rates. The protein-based coatings (NaCas and PP), both with and without surfactants showed the best results, suggesting that these matrices are more adequate supports for the BCA. CFAs also enhanced the efficacy of the BCA efficacy at controlling grey mold with respect to C. sake applied in water. NaCas and PP, and also some of the formulations based on S, exhibited the highest reduction values as regards to the incidence and severity of the infection. When the main properties of the coating forming dispersions and films were analyzed, it could be observed that the type of polymer, more than the presence or type of surfactant, greatly influenced the obtained values. The viability of C. sake on the different matrices was greatly reduced during storage at 25°C. However, protein-based coatings showed slightly higher counts. The coatings formed were not estimated to be tick and so they did not represent a relevant barrier to the gas exchanges of the fruit, although they were sufficient to improve the performance of C. sake as BCA. The physical stability of the different BCPs based on C. sake and starch derivatives (potato starch, pre-gelatinized potato starch and maltodextrins) was ensured below a water activity (aW) of 0.75 at room temperature, since BCPs were in a glassy state. However, the viability of C. sake at 20°C over time was greatly affected by the aW. Thus, whereas an aW ¿ 0.43 caused fast reductions in the viability of the BCA in all of the formulations, an aW ¿ 0.33 better preserved the viability of the yeast. This is a key factor since 0.33 is the aW value of a newly dried product and its moisturizing must be avoided in order to maintain its effectiveness in terms of cell viability. Nevertheless, 20°C was considered a non-adequate temperature since, even at low aW, a remarkable decline in viable cells was observed. However, cold storage of BCPs at 5°C allowed for a very good preservation of viable cells even after 6 months. A BCP based on maltodextrins as the main carrier was the formulation that showed the best potential to formulate C. sake in terms of the cell viability preservation and feasibility of in-field application due to its faster water solubilization. / [ES] El agente de biocontrol (ABC) Candida sake CPA-1 ha demostrado ser efectivo frente al hongo patogénico Botrytis cinerea, el agente causante de la podredumbre gris en muchas frutas. El objetivo de esta Tesis fue el desarrollo de productos de biocontrol (PBC) basados en este ABC y agentes formadores de recubrimiento (AFRs), con una buena estabilidad y efectividad frente a la infección fúngica. Se obtuvieron diversas formulaciones de AFRs, basadas en biopolímeros (hidroxipropilmetilcelulosa (HPMC), almidón de maíz (AM), caseinato sódico (NaCas) y proteína de guisantes (PG)), combinados con tensoactivos (ácido oleico (AO), Span 80 (S80) y Tween 85 (T85)). Todas ellas se analizaron en su capacidad para mejorar la adherencia, supervivencia y eficacia de C. sake en uvas. Se estudio también la funcionalidad de estas formulaciones como recubrimientos con y sin la incorporación de células de la levadura. Asimismo, se obtuvieron formulados secos basados en AFRs de bajo coste (derivados de almidón) y C. sake por secado en lecho fluido y su estabilidad física y microbiológica fue estudiada en función de su contenido en humedad. La aplicación de C. sake en combinación con AFRs permitió una mejora de la adherencia inicial de la levadura en la superficie de uvas y también una mayor supervivencia. Los recubrimientos basados en proteínas (NaCas y PG) con y sin tensoactivos mostraron los mejores resultados, sugiriendo que estas matrices son soportes más adecuados para el ABC. Los AFRs también mejoraron la efectividad del ABC en el control de la podredumbre gris en comparación con C. sake aplicada con agua. NaCas y PG, así como algunas de las formulaciones basadas en AM, dieron lugar a los valores más elevados de reducción de la incidencia y severidad de la infección. Cuando se analizaron las principales propiedades de las dispersiones formadoras de recubrimiento y las películas, se pudo observar que el tipo de polímero, más que la presencia o tipo de tensoactivo, afectó considerablemente los valores obtenidos. La viabilidad de C. sake en las distintas matrices se vio muy afectada durante su almacenamiento a 25°C. Sin embargo, los recubrimientos a base de proteínas mostraron recuentos ligeramente superiores. El espesor estimado de los recubrimientos formados en las uvas fue muy bajo, por lo que éstos no supusieron un efecto barrera relevante para los intercambios de gases de la fruta, aunque fueron suficientes para mejorar la función de C. sake como ABC. La estabilidad física de los diferentes PBCs basados en C. sake y derivados de almidón (almidón de patata, almidón de patata pregelatinizado y maltodextrinas) quedó asegurada a actividades de agua (aW) por debajo de 0.75 a temperatura ambiente, ya que los PBCs se encontraban en estado vítreo. Sin embargo, la viabilidad de C. sake a 25°C durante el tiempo se vio altamente disminuida. Mientras valores de aW ¿ 0.43 causaron rápidas reducciones en la viabilidad del ABC en todas las formulaciones, valores de aW ¿ 0.33 preservaron mejor la viabilidad de la levadura. Esto es un factor clave ya que 0.33 es el valor de aW de los productos recién obtenidos, por lo que su hidratación debe evitarse para mantener su efectividad en términos de viabilidad celular. No obstante, 20°C no fue considerada una temperatura de conservación adecuada ya que, incluso a valores bajos de aW, se observó un marcado descenso en el número de células viables. Por su parte, el almacenamiento en frío a 5°C permitió un muy buen mantenimiento de células viables, incluso tras 6 meses de almacenamiento. El PBC basado en maltodextrinas como soporte principal fue el formulado que mostró el mejor potencial para formular C. sake en términos de mantenimiento de la viabilidad celular y practicidad para su aplicación en campo, debido a su rápida solubilidad en agua. / [CA] L'agent de biocontrol (ABC) Candida sake CPA-1 ha demostrat s'efectiu enfront del fong patogènic Botrytis cinerea, l'agent causal de la podridura grisa en moltes fruites. L'objectiu d'aquesta Tesi va ser el desenvolupament de productes de biocontrol (PBC) basats en aquest ABC i agents formadors de recobriment (AFRs), amb una bona estabilitat i efectivitat enfront de la infecció fúngica. Es van obtindre diverses formulacions d'AFRs, basades en biopolímers (hidroxipropilmetilcelulosa (HPMC), midó de dacsa (M), caseinat sòdic (NaCas) i proteïna de pésol (PP)), combinats amb tensoactius (acid oleic (AO), Span 80 (S80) i Tween 85 (T85)). Totes elles van ser analitzades en la seua capacitat per a millorar l'adherència, supervivència i eficàcia de C. sake en raïm. La funcionalitat com a recobriments d'aquestes formulacions també va ser estudiada amb i sense la incorporació de cèl·lules del llevat. Tanmateix, es van obtindre formulats secs basats en AFRs de baix cost (derivats de midó) i C. sake per assecat en llit fluiditzat i la seua estabilitat física i microbiològica va ser estudiada en funció del seu contingut en humitat. L'aplicació de C. sake en combinació amb AFRs va permetre una millora de l'adherència inicial del llevat sobre la superfície de raïm i també una major supervivència. Els recobriments basats en proteïnes (NaCas i PP) amb i sense tensoactius van mostrar els millors resultats, suggerint que aquestes matrius són suports més adequats per a l'ABC. Els AFRs també van millorar l'efectivitat de l'ABC en el control de la podridura grisa en comparació amb C. sake aplicada amb aigua. NaCas i PP, així com algunes de les formulacions basades en M, van donar lloc als valors més elevats de reducció de la incidència i severitat de la infecció. Quan les principals propietats de les dispersions formadores de recobriment i pel·lícules van ser analitzades, es va poder observar que el tipus de polímer, més que la presència o tipus de tensoactiu, va afectar considerablement els valors obtinguts. La viabilitat de C. sake en les diferents matrius es va veure molt afectada durant el seu emmagatzemament a 25°C. No obstant això, els recobriments a base de proteïnes van mostrar recomptes lleugerament superiors. La grossària estimada dels recobriments formats en el raïm va ser molt baixa, per la qual cosa aquests no van suposar un efecte barrera rellevant per als intercanvis de gasos de la fruita, encara que van ser suficients per a millorar la funció de C. sake com ABC. L'estabilitat física dels diferents PBCs basats en C. sake i derivats de midó (midó de creïlla, midó de creïlla pregelatinitzat i maltodextrines) va quedar assegurada a activitats d'aigua (aW) per davall de 0.75 a temperatura ambient, ja que els PBCs es trobaven en estat vitri. No obstant això, la viabilitat de C. sake a 25°C durant el temps es va veure altament afectada. Mentre que valors d'aW ¿ 0.43 van causar ràpides reduccions en la viabilitat de l'ABC en totes les formulacions, valors d'aW ¿ 0.33 an preservar millor la viabilitat del llevat. Açò és un factor clau ja que 0.33 és el valor d'aW dels productes acabats d'obtindre, per la qual cosa la seua hidratació ha d'evitar-se per a mantindre la seua efectivitat en termes de viabilitat cel·lular. No obstant això, 20°C va ser considerada una temperatura no adequada ja que, fins i tot a valors d'aW baixos, un marcat descens en el nombre de cèl·lules viables va ser observat. Per la seua banda, l'emmagatzemament en fred a 5°C va permetre un molt bon manteniment de cèl·lules viables, fins i tot després de 6 mesos. El PBC basat en maltodextrines com a suport principal va ser el formulat que va mostrar el millor potencial per a formular C. sake en termes de manteniment de la viabilitat cel·lular i practicitat per a la seua aplicació en camp, degut a la seua ràpida solubilitat en aigua. / Marín Gozalbo, A. (2016). Coating forming agents as carriers of the biocontrol agent Candida sake with antifungal effect against Botrytis cinerea on grapes [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/71678 / Compendio

Vitis vinifera L.(Vv), the European cultivated grapevine is one of the most worldwide important crops but is highly susceptible to the necrotrophic fungus Botrytis cinerea (Bc), the causal agent of bunch rot (BR) disease. In grapevine, as well as in other fruit species, it has been described a primary infection by Bc at full bloom, followed by a quiescence period during the berry development, and the fungal egression after veraison reaching the maximum at harvest. Today, this important disease is mainly controlled by massive use of fungicides, which are applied at different developmental stages that happen to be critical during the grapevine-Bc interaction. During the contact, the fungus must overcome several barriers from the host, which protect it from the pathogen attack and might be also modulated or activated due to the presence of the pathogen itself. The cuticle and the cell wall (CW) represent the first barriers from the plant encountered by the pathogen. To successfully colonize the plant tissue, Bc possesses several virulence factors, and CW modifying enzymes (CWMEs) are part of them. On the other hand, the regulated activity of the CWMEs, expressed both by the host and the pathogen, could alter the plant CW composition and porosity, therefore facilitating, or limiting the penetration of the fungus. Among the CWMEs, Pectin methylesterases (PMEs) regulate the degree of methyl esterification of the pectin homogalacturonan, also modifying the epitopes for the activity of other CWMEs such as polygalacturonases (PGs) and pectate lyases (PLs) by whose action, pectin becomes more susceptible to degradation and the CW more accessible by the pathogen. Previous works both in Arabidopsis thaliana and in crop species identified several PME genes with an altered expression in response to pathogens. A previous report characterizing Atpme17 mutant lines has highlighted a role of AtPME17 in the resistance response to Bc in contrast with the opposite role of AtPME3, suggesting that PME genes could have a completely different action during Bc response depending on the isoform involved. In this context, the main objective of the project was to identify new strategies for Bc BR-control, and specifically i) to identify candidate genes involved in the response to Bc, whose inactivation/overexpression would lead to Bc resistant plants and ii) to set up a molecular method to monitor the Bc load in the field and therefore implement a more sustainable control of the pathogen. To further understand the effects of the Bc primary infection in grapevine flowers at CW level, two contrasting genotypes (Souvignier gris (SG) and Teroldego (TE)) in their resistance to the fungus were considered. An artificial inoculation of different biological replicates, in vase maintained, was performed at full bloom, in controlled conditions, and samples were collected at 24 hours post-inoculation (hpi) with the fungus and post-treatment with the respective control, for the following RNA-seq analysis and biochemical characterization of PME activity and CW composition in the two genotypes before and upon infection. The Bc load was estimated in the flowers using qPCR and as expected, a higher biomass of Bc was found in TE, the susceptible cultivar, than in SG, the resistant one. The analysis of CW composition, PME activity and degree of pectin methyl-esterification, both in treated and control flowers, showed significant differences between the two genotypes, in particular SG showed a significant induction of PME activity with respect to the control, evidence not present in the susceptible genotype. The RNAseq analysis on the same samples showed a total of 4800 genes modulated, out of which 3064 are only modulated in TE, 739 only in SG and a common group of 997 genes. Regardless of the cultivar, upon infection there was a total 2919 genes upregulated vs 1909 genes downregulated. A gene set enrichment analysis (GSEA) indicated several over-represented categories upon infection, including response to pathogens and biosynthesis of secondary metabolites, with a general down-regulation of those genes related to CW organization and pectin modification (CWMEs), mostly in the resistant genotype. Within the down-regulated CWMEs, Pectin methylesterase (PME) genes were found highly represented. Unlike, a larger gene set, in many cases with a higher fold-change of induction, was identified in TE respect to SG. This is the case of genes involved in the defense response and its regulation, and in the modification/reinforcement of the cell wall, therefore attesting for an initial tentative by the susceptible genotype to counteract the pathogen, although at the end without success. This was also the case of the seven VviPME genes previously highlighted by the in-silico co-expression analysis and therefore of VviPME10, the gene with the highest homology to AtPME17. Among the regulators, one WRKY factor (VviWRKY3), known to be related to defense response in grapevine mediated by stilbene synthesis, was also further characterized as putative regulator of VviPME10, whose promoter hosts more than one several predicted binding sites for VviWRKY3. Indeed, luciferase assay results indicate a significant activation of VviPME10 promoter by VviWRKY3 factor. Parallelly, the genome-wide analysis of the last structural annotation of the grapevine genome assembly allowed us to identify 62 VviPME gene members, 15 more than a previous report, and manually curate the gene structure for 39 of them. Then, to corroborate the idea of the role of the CW, and in particular of PME activity, in the grapevine response to the fungus, an in silico co-expression analysis of the 62 VviPME members, considering the publicly available RNA-seq experiments related to grapevine-Bc interactions and the RNAseq experiment conducted in this project, was performed. The analysis highlighted a group of seven genes (VviPME1, VvPME9, VviPME10, VviPME11, VviPME12, VviPME13 and VviPME54) with significant induction upon Bc infection, five of them (VviPME8, VviPME9, VviPME10, VviPME11, and VviPME54) located in the same chromosome (chr06). VviPME10 showed the highest homology and was found to be phylogenetically close to the Arabidopsis thaliana PME17 gene, suggesting being considered as its putative orthologue. Afterward, Therefore, considering the increased VviPME10 expression upon infection, and the reported effect of AtPME17 in A. thaliana, VviPME10 was selected as a potential candidate to study its role in grapevine. In this regard, two strategies were adopted, i. VviPME10 knock-out (KO) with CRISPR/Cas9 and ii. VviPME10 overexpression (OE, under CaMV35S promoter) through embryogenic callus transformation of the grapevine cultivar ‘Sugraone’ mediated by Agrobacterium tumefaciens. More than 100 embryos developed, and around 20 plantlets per transformation were analyzed to check the presence of the transgenic construct. Then, the mutation profile, in the case of KO lines, and the expression analysis of the transgene, in the case of OE lines, were carried out to select the appropriate lines to acclimatize. OE lines were also tested for VviPME10 activity. A total protein extract was obtained from the leaves of the lines, showing a higher protein activity compared to the control, and indicating the functionality of the enzyme. Unfortunately, grapevine OE lines couldn’t be analyzed for their response to Bc, while KO lines showed a significantly larger lesion area when compared to the control at 5 days post fungal inoculation (dpi). However, the effect of VviPME10 overexpression upon Bc infection was evaluated also in Nicotiana benthamiana VviPME10-OE lines, generated in parallel. At 3 dpi a significant reduction was observed in the lesion area compared to the control. These results suggest that pectin modification, mediated by VviPME10, plays an important role in the grapevine response to Bc, in particular it seems to behave more like a resistance gene than a susceptibility one. For this reason, it could be considered as a valuable target to improve resistance to Bc in susceptible grapevine varieties. Finally, a molecular method for Bc detection, based on quantitative RT-PCR assays, was set up and applied to estimate the Bc load in field conditions. Although the method allowed the successful detection of the presence of the fungus in samples at different developmental stages, from two V. vinifera cultivars, in different vineyards, the lack of environmental conditions for the development of the disease might have impaired the correlation between detection and the development of the disease. Nonetheless, the method represents a good alternative for monitoring the Bc load in the field at the early season, to predict the BcBR severity at harvest and eventually apply the disease management protocols based on the real need.