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Embalaje con bolsa de polietileno de permeabilidad restringida y su efecto en la conservación de uva de mesa var. crimson seedlessArratia París, Marcela Bernardita January 2005 (has links)
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Aislados chilenos de Botrytis cinerea resistentes a Iprodione : niveles de virulencia y caracterización del gen bos1Araneda Rubio, María José January 2011 (has links)
Memoria para optar al título profesional de
Ingeniero Agrónomo / Con el fin de evaluar la virulencia y características genéticas de aislados chilenos
de Botrytis cinerea con distinta sensibilidad a iprodione, se seleccionaron nueve
aislados altamente resistentes (EC50: 1,35 – 2,47 μg·mL-1) y cuatro aislados
sensibles (EC50: 0,26 – 0,31 μg·mL-1).
La virulencia se evaluó en plántulas de pepino (Cucumis sativus L.) mediante
inoculación de discos de agar con micelio del hongo de tres días de edad y en bayas
de vid (Vitis vinífera L.) Thompson Seedless con distinto grado de madurez (7°, 14°
y 17° Brix), mediante inoculación de una suspensión conidial (equivalente a 2·105
conidias·mL-1). Posterior a la inoculación las plántulas de pepino se mantuvieron a
20-25°C y 100% de humedad relativa y las bayas de vid en cámaras de crecimiento
a 20°C. La evaluación se realizó mediante medición del diámetro de la lesión en la
zona de inoculación luego de 96 y 72 horas en hojas de pepino y bayas de vid,
respectivamente. Los resultados obtenidos fueron sometidos a un ANDEVA simple
y en las dos pruebas de virulencia realizadas los aislados de Botrytis cinerea
resistentes y sensibles presentaron niveles de agresividad similares.
La caracterización genética de los aislados se realizó amplificando mediante PCR el
gen bos1 asociado a resistencia de Botrytis cinerea a dicarboximidas, utilizando 5
pares de partidores específicos. Posteriormente los productos de PCR purificados
fueron secuenciados por Macrogen USA Corp.
El análisis de las secuencias nucleotídicas de los nueve aislados resistentes detectó
un cambio aminoacídico en la posición T1259A de la proteína. En ocho de éstos
fueron detectadas las mutaciones I365N y R104Q y en el aislado restante un
cambio en la posición I365S y una sustitución en la posición L849V. Esta última
sustitución no ha sido descrita previamente desconociéndose su implicancia en el
nivel de resistencia. En ninguno de los cuatro aislados sensibles secuenciados, el
gen bos1 presentó mutaciones asociadas a resistencia a dicarboximidas. / In order to evaluate the virulence and genetic characteristics to Chilean isolates
Botrytis cinerea of with different sensitivity to iprodione, were selected nine highly
resistant isolates (EC50: 1, 35 to 2.47 μg·mL-1) and four susceptible isolates (EC50:
0.26 to 0.31 μg·mL-1).
The virulence was evaluated inoculating cucumber seedlings (Cucumis sativus L.)
with three days old Botrytis cinerea mycelium and different ripe (7 °, 14 ° 17 °
Brix) Thompson Seedless grape berries (Vitis vinifera L.) with a conidial
suspension (2·105 conidia·mL-1).
Cucumber seedlings were kept at 20-25°C and relative humidity 100% and grape
berries in growth chambers at 20°C. The evaluation was done measuring the lesion
diameter after 96 and 72 hours in cucumber leaves and grape berries, respectively.
The results were subjected to a simple ANOVA and in both virulence tests carried
out, the sensitive and resistant Botrytis cinerea isolates showed similar levels of
aggressiveness.
The genetic characterization was performed by PCR amplification of the
dicarboximide resistance associated Botrytis cinerea bos1 gene, by means of five
specific primers. The purified PCR product was sequenced by Macrogen USA
Corp.
By means of sequences analysis the bos1 gene nucleotide, in all the nine resistant
isolates was detected an amino acid change at the T1259A protein position. The
I365N and R104Q mutations were detected in eight of the isolates and in the other
isolate a change in the position I365S and a substitution at position L849V. The
latter substitution has not been previously detected and it implications on resistance
is unknown. The bos1 gene showed no mutations associated to the resistance to
dicarboximides in any one of the four susceptible isolates sequenced.
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Caracterización genotípica y fenotípica de las poblaciones de Botrytis cinerea predominantes en Actinidia deliciosa de un huerto de la VII Región / Genotypic and phenotypic characterization of Botrytis cinerea predominate populations in Actinidia deliciosa in an orchard of Region VIICamus Soto, Natalia Macarena January 2014 (has links)
Memoria para optar al título profesional de Ingeniera Agrónoma / Se estudió la dinámica poblacional de Botrytis cinerea (Bc) en el cultivo del kiwi en Chile, caracterizando genética y fenotípicamente un número representativo de aislados recuperados desde flores y frutos sintomáticos colectados en pre y poscosecha en un huerto del Valle Central. En total se seleccionaron completamente al azar 276 aislados monospóricos, determinándose la correspondencia genotípica mediante PCR-dúplex y el uso de partidores específicos (amplificación de secuencias transponibles Boty y Flipper). Posteriormente, 10 aislados de cada genotipo identificado se caracterizaron fenotípicamente con respecto a: 1) aspecto morfológico (miceliar/esclerocial), 2) capacidad formadora de esclerocios, 3) nivel de esporulación a 20ºC (Agar Malta) y 4) crecimiento miceliar a 15, 20 y 25ºC (agar malta); evaluándose además la virulencia de éstos mismos aislados en: a) bayas de vid cv. Thompson Seedless de 16,5º Brix de madurez, inoculadas con y sin herida y mantenidas en incubación a 20ºC por 96 horas (nº de bayas = 150/genotipo; 25 por repetición y condición) y b) en frutos de kiwi cv. Hayward (7-8º Brix) inoculados solo con heridas (nº de frutos = 100/genotipo; 25 por repetición), incubados a 20ºC y 0ºC por 17 y 60 días, respectivamente.
Los resultados obtenidos señalan a vacuma como el genotipo predominante en floración (36%) y poscosecha (69.2%); transposa se presenta en menor proporción durante floración (20%), aumenta en cosecha (84%), y en poscosecha solo corresponde a un 7,7%. Los aislados tipo flipper, se presentaron en una baja a moderada frecuencia en todos los estadios fenológicos o periodos analizados, con excepción de fruto recién formado (24%); finalmente los aislados tipo boty solo se recuperaron en una alta proporción durante floración (32%), no detectándose en poscosecha. Estos resultados señalan que la composición de la población de Bc en kiwi difiere notoriamente de la descrita en uva de mesa de Chile, en la cual transposa predomina en todos los estadios fenológicos, y vacuma sólo se presenta en una muy baja proporción y casi exclusivamente en floración.
Fenotípicamente, los aislados se comportaron de manera similar independientemente de la correspondencia genotípica (p > 0,05). En general, al evaluar la virulencia en bayas de vid a 20°C, los mayores diámetros de lesión en la condición con herida, fueron presentados por los aislados flipper (p < 0,05), pero sin diferencias con los demás genotipos en la modalidad sin herida (p > 0,05). En kiwis incubados a 20°C, aunque no se detectaron diferencias significativas entre aislados Bc de los distintos genotipos, vacuma fue el más virulento; en cambio a 0ºC, los aislados vacuma y boty fueron más agresivos diferenciándose significativamente de los aislados transposa y flipper (p < 0,05).
Los resultados obtenidos permiten concluir la existencia de una posible asociación entre la composición genotípica de las poblaciones de Bc presentes en kiwi y el nivel de daño por pudriciones del patógeno en poscosecha. / The population dynamics of Botrytis cinerea (Bc) on a kiwifruit crop in Chile was studied by genetic and phenotypic characterization of a representative number of isolates from symptomatic flowers and fruits collected at pre- and postharvest in an orchard located in the Central Valley. In total, 279 randomly selected monosporic isolates were used, with the genotypic correspondence being determined by the duplex PCR reaction and the use of specific primers (amplification of transposable sequences Boty and Flipper). Later, 10 isolates of each identified genotype were phenotypically characterized with respect to: 1) morphological aspect (mycelial/sclerotical); 2) sclerotium-forming ability; 3) sporulation level at 20°C (malt agar); and 4) mycelial growth at 15, 20 and 25°C (malt agar). In addition, the virulence of the same isolates was evaluated in: a) ‘Thompson Seedless’ grape berries having 16,5°Brix of maturity, inoculated with and without wound and kept under incubation at 20°C for 96 hours (N° of berries = 150/genotype; 25 per replication and condition); and b) ‘Hayward’ kiwifruits (7-8°Brix) inoculated just with wounds (N° of fruits = 100/genotype; 25 per replication), incubated at 20 and 0°C for 17 and 60 days, respectively.
The results obtained show vacuma as the predominat genotype (69.2%); transposa occured in a smaller proportion during bloom (20%), it increased at harvest time (84%), and at postharvest it only reached 7.7%. Flipper-type isolates occurred in a low-to-moderate frequency at all the phonological stages or periods analyzed, excepting newly-formed fruit (24%), while boty-type isolates were recovered in high proportion only during bloom (32%), not being detected at postharvest. These results indicate that B. cinerea population make-up differs markedly from that described in table grapes in Chile where transposa prevails at all the phenological stages, and vacuma occurs just in a very low proportion and almost exclusively at bloom.
Phenotypically, the isolates behaved similarly irrespective of the genotypic correspondence (p >0.05). When evaluating the virulence, the greatest lesion diameters in wounded grape berries were shown by flipper isolates (p <0.05), but with no differences with the other genotypes in the unwounded group (p >0.05). In kiwifruit incubated at 20°C, although no significant differences were detected among Bc isolates of the different genotypes, vacuma was always the most virulent; conversely, at 0°C vacuma and boty isolate were more aggressive and differentiated significantly from the transposa and flipper isolates (p< 0.05).
The results obtained suggest the existence of a possible association between the genotypic composition of Bc populations occurring in kiwifruit and the level of damage from rots caused by the pathogen at postharvest.
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Uso de ácido giberélico para disminuir la compactación de racimo en variedad zinfandel / Use of gibberellic acid to decrease cluster compactness in zinfandel grapeCarmona León, Bruno René January 2017 (has links)
Memoria para optar al Título Profesional de Ingeniero Agrónomo / Durante la temporada 2013-2014, se realizó un ensayo de campo en la variedad de vid Zinfandel, para determinar el efecto de la aplicación de diferentes concentraciones de ácido giberélico, en tres estados fenológicos, sobre la compactación de racimos y la incidencia de pudrición gris causada por Botrytis cinerea. Para el ensayo se utilizaron racimos en tres estados fenológicos (prefloración, 25% de floración y 50% de floración) a los que se les aplicó ácido giberélico (GA3) en concentraciones de 0, 10, 20 y 40 mg٠L-1. Se realizó un total
de 12 tratamientos que resultaron de la combinación de los factores estado fenológico y concentración de ácido giberélico. Se evaluaron diversas características del racimo, incluyendo longitud, compactación, peso y volumen de racimo, peso de bayas, número de bayas, contenido de sólidos solubles y la incidencia y severidad de la pudrición gris. Se observó una tendencia a la disminución de la compactación a medida que aumentó la concentración de ácido giberélico, reflejada en el índice de compactación, aunque en la escala de compactación no se observaron diferencias. Al inicio de las evaluaciones se observaron mayores longitudes de raquis con el aumento de las concentraciones de ácido giberélico, pero estas diferencias desaparecieron a cosecha. Los tratamientos no mostraron diferencias significativas en la incidencia y severidad de pudrición gris.
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Sensibilidad in vitro E in vivo de cepas de Botrytis cinerea y Penicillium expansum aisladas de manzanas en poscosecha a : tiabendazol, fludioxonil y productos químicos de origen natural / In vitro and in vivo sensitivity of Botrytis cinerea and penicillium expansum strains isolated from apples in postharvest to: thiabendazole, fludioxonil and natural chemical productsBarcos Muñoz, Javiera Paz January 2011 (has links)
Memoria para optar al título profesional de: Ingeniera Agrónoma / Uno de los factores que tiene mayor incidencia en la condición de la fruta son las enfermedades de poscosecha, conocidas comúnmente como “pudriciones”. Para el caso de manzanas las más frecuentes son las causadas por Penicillium expansum y Botrytis cinerea. Las aplicaciones de fungicidas sintéticos en poscosecha son una de las estrategias más utilizadas para su control; sin embargo, su uso continuado y prolongado ha generado una disminución de su efecto protector. Tiabendazol ha sido utilizado de manera prolongada como fungicida en el control de dichas enfermedades, pero su uso ha sido limitado por el desarrollo de cepas fitopatógenas resistentes. En el año 2004 fludioxonil fue registrado en Estados Unidos de Norte América para tratamientos de poscosecha en manzanas, siendo altamente efectivo en el control de los patógenos que causan dichas pudriciones incluyendo cepas resistentes a tiabendazol. También se han propuesto otras alternativas de control, como por ejemplo los compuestos antifúngicos naturales. El objetivo del presente estudio fue evaluar la sensibilidad de cepas de Botrytis cinerea y Penicillium expansum aisladas de manzanas, a los fungicidas tiabendazol (Tecto® 500 SC), fludioxonil (Scholar® 50 WP) y productos de origen natural (BC-1000®, Lonlife plus® SC y Biorend® SC). Se determinó in vitro la concentración media efectiva (EC50) y concentración mínima inhibitoria (CMI) la para micelio y conidias a cada fungicida, posteriormente se evaluaron las dosis comerciales de cada producto en condiciones in vivo. Un 10% y un 48% de las cepas de Botrytis cinerea y de Penicillium expansum respectivamente, resultaron ser resistentes a tiabendazol. Fludioxonil fue efectivo en la inhibición de ambos patógenos con valores de EC50 promedio para conidias de 0,02 μg·mL-1 para Botrytis cinerea y de 0,29 μg·mL-1 para Penicillium expansum. Los productos de origen natural presentaron resultados intermedios inhibiendo algunas de las cepas evaluadas, excepto la formulación de BC-1000® utilizada, que no inhibió a ninguno de los dos patógenos. En los ensayos in vivo, el mayor efecto se obtuvo con fludioxonil, y tiabendazol resultó efectivo solo contra las cepas sensibles de ambos patógenos. Las dosis comerciales de los productos naturales utilizados ejercieron un control relativo de ambos patógenos, salvo BC-1000® que no logró efecto de control alguno sobre ambos patógenos. / One of the factors that most affect the condition of the fruit are postharvest diseases, commonly known as "fruit rots”. The most common in apples are caused by Penicillium expansum and Botrytis cinerea. The application of synthetic fungicides in postharvest is one of the strategies used for their control; however, continued use of agrochemicals has led to a decrease in their protective effect. Thiabendazole as a fungicide has been used for a long time in the control of these diseases, but its use has been limited by the development of resistant phytopathogenic strains. In 2004 Fludioxonil was registered in United States of America for use in apples in postharvest treatments being highly effective in controlling these pathogens including resistant strains to thiabendazole. Natural antifungal compounds have also been proposed for postharvest fruit rot control. The aim of this study was to evaluate to sensitivity of Botrytis cinerea and Penicillium expansum strains isolated from apples, the fungicides thiabendazole (Tecto ® 500 SC), fludioxonil (Scholar ® 50 WP) and natural products (BC-1000 ®, Lonlife Plus® SC and Biorend® SC). EC50 and MIC were in vitro calculated for inhibition of mycelium growth and conidial germination for each fungicide, while the commercial dose of each product was in vivo evaluated. 10% and 48% of strains of Botrytis cinerea and Penicillium expansum respectively, were resistant to thiabendazole. Fludioxonil was effective in both pathogens with EC50 values of 0.02 average conidia μg·mL-1 to Botrytis cinerea and 0.29 μg·mL-1 for Penicillium expansum. The natural products showed intermediate results inhibiting some of the strains, except for the formulation of BC-1000 ® used, that did not inhibit any of the two pathogens. Fludioxonil was the most effective active ingredient reducing in vivo both decays, although thiabendazole was only effective against susceptible strains of both pathogens.
BC-1000® formulation commercial doses of natural products used exerted a relative control of both pathogens.
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Impact du déficit hydrique sur les réponses de défense et la sensibilité de la vigne à Botrytis cinerea : rôle de la dégradation des polyamines. / Impact of water deficiency on defense responses and susceptibility of grapevine to Botrytis cinerea : Role of polyamine oxidationHatmi, Saloua 18 December 2013 (has links)
La vigne est sujette à de nombreuses contraintes biotiques et abiotiques face auxquelles elle devra optimiser ses stratégies de défense, en favorisant parfois des interconnections entre les réponses adaptatives au stress abiotique et la gestion de la réponse immune face à un pathogène. Dans cette étude nous avons évalué l'effet du stress hydrique (par privation d'eau) sur différentes réactions adaptatives au stress, mais aussi sur des réponses de défense et sur la sensibilité des feuilles et des baies de la vigne à Botrytis cinerea. Ces réactions ont été suivies en utilisant des boutures végétatives de deux cépages : cv. Meski (MSK), tolérant à la sécheresse et cv. Chardonnay (CHR), sensible. La relation entre les réponses au stress hydrique et la réponse immune a également été recherchée au moyen de feuilles de vitroplants et de baies isolées de Chardonnay exposées à des osmotica : le polyéthylène glycol (PEG) et une forte concentration en saccahrose (SUC). Les résultats montrent que le stress hydrique/osmotique conduit à des modifications physiologiques et biochimiques importantes dans les feuilles et les baies mûres de vigne. L'amélioration de la tolérance chez MSK est associée à une faible inhibition de l'activité photosynthétique, une altération du profil des acides aminés et un catabolisme actif des polyamines (PAs) comparé au CHR. Ces résultats suggèrent un rôle potentiel des déviations métaboliques observées dans les processus de tolérance de la vigne au stress osmotique. La tolérance du MSK au déficit hydrique est également corrélée à une forte induction des réponses de défense accumulation de resvératrol et e-viniférine, expression de certains gènes de défense dont STS, Gluc (PR-2), Chit-4c (PR-3) et PR-5 dans les feuilles, ainsi qu'à une faible sensibilité à B. cinerea.Ces résultats suggèrent un lien étroit entre la tolérance au déficit hydrique et la capacité de la vigne à exprimer davantage ses mécanismes de défense et à résister mieux à B. cinerea. Des expériences pharmacologiques ont montré qu'en situation de stress hydrique/osmotique, le catabolisme des PAs, via des diamine- et PA-oxydases, est impliqué dans la régulation de l'homéostasie des PAs et de l'expression des réactions de défense. L'application du stress osmotique avant l'infection des feuilles par B. cinerea potentialise l'accumulation des PAs en réduisant fortement leur dégradation. Ces effets sont corrélés à une réduction de l'amplitude des réponses de défense après infection, et à une sensibilité accrue à B. cinerea. Ces résultats rendent compte (1) de l'importance des stress abiotiques dans la régulation de la réponse immune chez la vigne et sa résistance à B. cinerea et (2) du fait que le niveau des réponses de défense osmo-induites et la résistance au pathogène sont tributaires, au moins en partie, de certains mécanismes adaptatifs au stress, comme c'est le cas ici des voies de dégradation des polyamines. / Grapevine is often exposed to both biotic and abiotic stresses, and it will optimize defense strategies by favoing sometimes the cross-talk between adaptive responses to abiotic stress and immune response against pathogen challenge. In this study we evaluated the effect of water stress (by withholding water) on different adaptive responses, and also on defense responses and sensitivity of grapevine leaves or berries to Botrytis cinerea. These reactions were monitored using vegetative cuttings of two varieties: Meski (MSK) a drought tolerant cultivar and Chardonnay (CHR) as a sensitive one. The relationship between the responses to water stress and the immune response was also assessed using detached leaves from vitroplantlets and detached ripe berries from Chardonnay exposed to osmotic agents: polyethlene glycole (PEG) and a high concentration of sucrose (SUC). The results show that water/osmotic stress leads to significant physiological and biochemical changes in grapevine leaves and ripe berries. The improved tolerance of MSK to drought is associated with a weak inhibition of photosynthetic activity, altered amino acid profile and an activation of polyamine (PA) catabolism, compared to the sensitive plant CHR. These results suggest a potential role of metabolic deviations observed in the process of osmotic stress olerance. MSK tolerance to water deficit is also correlated with a strong induction of defense responses, such as accumulation of resveratrol and e-viniferin, enhanced expression of defense-related genes, including STS , Gluc (PR-2), Chit-4c (PR-3) and PR-5, and a low susceptibility of leaves to B. cinerea. These results suggest a close connection between water stress tolerance and the ability of grapevine to express more their defense mechanisms and then to resist better to the pathogen B. cinerea. Pharmacological experiments showed that experiencing water/osmotic stress, PA oxidation through diamine- and PA-oxidases is involved in the regulation of PA homeostasis and the expression of defense reactions in both leaves and berries. The application of osmotic stress before leaf infection by B. cinerea potentiates PA accumulation probably by reducing PA degradation. These effects are correlated with a reduction of defense responses after B. cinerea infection, as well as to an increased susceptibility to B. cinerea.These results highlight (1) the importance of abiotic stress in regulating the immune response in grapevine plants and resistance to B. cinerea and (2) that the level of defense responses induced by osmotic and the resistance of grapevine to the pathogen are dependent, at least in part, on some adaptive mechanisms to stress, as it is the case here for polyamine degradation pathways.
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Caracterização de lacase de Peniophora cinerea e estudo do potencial de aplicação biotecnológica / Characterization of Peniophora cinerea laccase and study of the potential for biotechnological applicationsMoreira Neto, Sergio Luiz 16 March 2012 (has links)
No presente estudo, fungos ligninolíticos isolados de ecossistemas brasileiros foram avaliados quanto a capacidade de descolorir corantes reativos e, a linhagem Peniophora cinerea, selecionada, teve seu sistema ligninolítico caracterizado. As lacases deste fungo foram avaliadas para duas aplicações: descoloração de efluentes têxteis e auxílio na hidrólise enzimática do bagaço de cana-de-açúcar. Lacase foi a única enzima ligninolítica extracelular detectada nos culivos de P. cinerea. A produção de lacase por P. cinerea foi avaliada em meios sintético e complexo, de forma a obter melhores níveis de produção da enzima. Em meio complexo (milhocina 0,5%, sacarose 0,5% e cobre como indutor de lacase) P. cinerea produziu cerca de aproximadamente 1000 U/l de lacase. Em meio sintético, contendo xilidina e tween 80, P. cinerea imobilizado (em esponja de poliuretano) produziu 3505 U/l de lacase em cultivo em Erlenmeyer e somente 150 U/l em reator de tanque com agitação por pás. Lacases produzidas por P. cinerea foram purificadas e caracterizadas. O sobrenadante do meio de cultivo foi aplicado em coluna de troca aniônica, DEAE-Sepharose CL-6B, e três picos com atividade de lacase foram observados e aplicados separadamente em Mono Q (coluna de troca aniônica). Oito isoenzimas de lacase, com massas entre 30 e 70 kDa e ponto isoelétrico (pI) entre 3 e 6 foram reveladas em géis de eletroforese em condições desnaturantes e não desnaturantes e por focalização isoelétrica (IEF). Algumas isoenzimas apresentaram alta termoestabilidade a 50ºC, pH ótimo entre 2,6 e 4,0 e Km para o ABTS entre 12,7 e 20 ?M, dependendo do tipo de lacase. Diferentes mediadores de lacases de P. cinerea foram avaliados (HBT, HBA, 2,3 DHBA, DOPAC, ABTS, siringaldeido, oxalato de sódio e Mn2+). O sistema composto por lacase, siringaldeído, oxalato de sódio e Mn2+ produziu a maior descoloração do Reactive Blue 19. As lacases foram utilizadas para descoloração de um efluente simulado, preparado com 100 mg/l de Reactive Red 271, hidrolisado em NaOH 2 M, e um efluente real, fornecido por uma indústria têxtil. O efluente simulado foi totalmente descolorido, em 24h, pelo extrato enzimático. A partir desta constatação, o efluente real foi tratado com os extratos de P. cinerea e mais de 60% de descoloração foi obtida com o uso de lacase e mediadores. Lacases produzidas por P. cinerea foram também utilizadas para hidrólise de bagaço de cana-de-açúcar. As amostras de bagaços foram obtidas de tratamentos com sulfito ácido, sulfito alcalino, ácido sulfúrico, clorito de sódio ou sem tratamento (in natura). Duas formas de tratamento foram avaliadas: tratamento simultâneo, ou seja, lacases foram adicionadas juntamente com um preparado comercial de celulases; ou num tratamento sequencial, em que a polpa do bagaço foi previamente tratada com lacase para depois ser hidrolisada com celulases. O tratamento simultâneo com lacase e celulase resultou em menor conversão de celulose, devido a possível inativação das celulases pela ação oxidativa de lacase. A aplicação sequencial das enzimas nos bagaços pré-tratados por sulfito ácido ou por sulfito alcalino favoreceu à hidrólise da celulose. Estudos futuros para tratamento sequencial de lacase à hidrólise de bagaço de cana devem considerar aspectos reacionais básicos: maior carga enzimática, temperatura, tempo de reação e mediadores. Lacases são excepcionalmente versáteis para aplicações biotecnológicas e a produção em larga escala integrará o uso da enzima em diferentes processos industriais. / In the present estudy, ligninolytic fungi isolated from Brazilian ecosystems were evaluated for the ability to decolorize reactive dyes and a selected strain of Peniophora cinerea had its ligninolytic system characterized. Laccases from this fungi were evaluated for two biotechnology applications: decolorization of textile effluents and enzymatic hydrolysis of sugarcane bagasse. P. cinerea produced an halotolerant laccase activity, unique feature with the data available in the literature and with potential for biotechnological applications, making it the object of our studies. Laccase was the only ligninolytic enzyme detected in the extracellular extract from P. cinerea. The production of laccase by P. cinerea was evaluated in synthetic and complex media, in order to obtain higher levels of enzyme production. In complex medium (corn steep liquor 0.5%, sucrose 0.5% and copper, as inducer of laccase) P. cinerea produced about 1000 U/l of laccase. In synthetic medium containing tween 80 and xylidine, P. cinerea immobilized in polyurethane foam produced 3505 U/l of laccase in culture in flasks; in STR reactor P. cinerea produced only 150 U/l. Laccases produced by P. cinerea were purified and characterized. The supernatant of the culture medium was loaded on an anion exchange column, DEAE-Sepharose CL-6B, and three peaks with laccase activity were detected and applied separately on a Mono Q column. Eight isoenzymes of laccases with molecular weigh between 30 and 70 kDa and isoelectric point (pI) between 3 and 6 were revealed on electrophoresis gels at denaturing, native conditions and isoelectric foccusing (IEF). Some isoenzymes showed high thermostability at 50°C, optimal pH between 2.6 and 4.0, and Km for the ABTS between 12.7 and 20 ?M., depending on the type of laccase. Different mediators for laccases of P. cinerea were evaluated (HBT, HBA, 2,3 DHBA, DOPAC, ABTS, syringaldehyde, sodium oxalate and Mn2+). The system laccase, syringaldehyde, sodium oxalate and Mn2+ produced the highest decolorization of Reactive Blue 19. Laccases produced by P. cinerea were used for decolorization of a simulated efluent prepared with 100 mg/l of Reactive Red 271 hydrolyzed with NaOH 2 M and a real effluent, provided by a textile industry. The simulated efluent was completely decolorized within 24 hours by the enzymatic extract. In face of that, the real effluent was evaluated for decolorization by the extracts of P. cinerea and more than 60% of decolorization was obtained by laccase-mediator system. Laccases produced by P. cinerea were also used to hydrolize the sugar cane bagasse. Sugarcane bagasse samples were obtained by acid sulfite, alkaline sulfite, sulfuric acid, sodium chlorite or untreated (in natura). Two forms of treatment were evaluated: simultaneous treatment, ie, laccases have been added along with a commercial cellulase complex for hydrolysis of sugarcane bagasse, or a sequential treatment, in witch bagasse was pre-treated with laccase followed by cellulases. Simultaneous treatment with both enzymes resulted in lower conversion of cellulose than sequential treatment due to possible inactivation of cellulases by the oxidative action of laccase. Future studies of sequential treatment with laccase and hydrolysis of sugarcane bagasse should consider additional basic concepts of reactions such as: increase of enzyme load, temperature, reaction time and mediators. Laccases are exceptionally versatile for biotechnological applications and the production in large scale integrate the use of the enzyme in different industrial processes.
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Caracterização de lacase de Peniophora cinerea e estudo do potencial de aplicação biotecnológica / Characterization of Peniophora cinerea laccase and study of the potential for biotechnological applicationsSergio Luiz Moreira Neto 16 March 2012 (has links)
No presente estudo, fungos ligninolíticos isolados de ecossistemas brasileiros foram avaliados quanto a capacidade de descolorir corantes reativos e, a linhagem Peniophora cinerea, selecionada, teve seu sistema ligninolítico caracterizado. As lacases deste fungo foram avaliadas para duas aplicações: descoloração de efluentes têxteis e auxílio na hidrólise enzimática do bagaço de cana-de-açúcar. Lacase foi a única enzima ligninolítica extracelular detectada nos culivos de P. cinerea. A produção de lacase por P. cinerea foi avaliada em meios sintético e complexo, de forma a obter melhores níveis de produção da enzima. Em meio complexo (milhocina 0,5%, sacarose 0,5% e cobre como indutor de lacase) P. cinerea produziu cerca de aproximadamente 1000 U/l de lacase. Em meio sintético, contendo xilidina e tween 80, P. cinerea imobilizado (em esponja de poliuretano) produziu 3505 U/l de lacase em cultivo em Erlenmeyer e somente 150 U/l em reator de tanque com agitação por pás. Lacases produzidas por P. cinerea foram purificadas e caracterizadas. O sobrenadante do meio de cultivo foi aplicado em coluna de troca aniônica, DEAE-Sepharose CL-6B, e três picos com atividade de lacase foram observados e aplicados separadamente em Mono Q (coluna de troca aniônica). Oito isoenzimas de lacase, com massas entre 30 e 70 kDa e ponto isoelétrico (pI) entre 3 e 6 foram reveladas em géis de eletroforese em condições desnaturantes e não desnaturantes e por focalização isoelétrica (IEF). Algumas isoenzimas apresentaram alta termoestabilidade a 50ºC, pH ótimo entre 2,6 e 4,0 e Km para o ABTS entre 12,7 e 20 ?M, dependendo do tipo de lacase. Diferentes mediadores de lacases de P. cinerea foram avaliados (HBT, HBA, 2,3 DHBA, DOPAC, ABTS, siringaldeido, oxalato de sódio e Mn2+). O sistema composto por lacase, siringaldeído, oxalato de sódio e Mn2+ produziu a maior descoloração do Reactive Blue 19. As lacases foram utilizadas para descoloração de um efluente simulado, preparado com 100 mg/l de Reactive Red 271, hidrolisado em NaOH 2 M, e um efluente real, fornecido por uma indústria têxtil. O efluente simulado foi totalmente descolorido, em 24h, pelo extrato enzimático. A partir desta constatação, o efluente real foi tratado com os extratos de P. cinerea e mais de 60% de descoloração foi obtida com o uso de lacase e mediadores. Lacases produzidas por P. cinerea foram também utilizadas para hidrólise de bagaço de cana-de-açúcar. As amostras de bagaços foram obtidas de tratamentos com sulfito ácido, sulfito alcalino, ácido sulfúrico, clorito de sódio ou sem tratamento (in natura). Duas formas de tratamento foram avaliadas: tratamento simultâneo, ou seja, lacases foram adicionadas juntamente com um preparado comercial de celulases; ou num tratamento sequencial, em que a polpa do bagaço foi previamente tratada com lacase para depois ser hidrolisada com celulases. O tratamento simultâneo com lacase e celulase resultou em menor conversão de celulose, devido a possível inativação das celulases pela ação oxidativa de lacase. A aplicação sequencial das enzimas nos bagaços pré-tratados por sulfito ácido ou por sulfito alcalino favoreceu à hidrólise da celulose. Estudos futuros para tratamento sequencial de lacase à hidrólise de bagaço de cana devem considerar aspectos reacionais básicos: maior carga enzimática, temperatura, tempo de reação e mediadores. Lacases são excepcionalmente versáteis para aplicações biotecnológicas e a produção em larga escala integrará o uso da enzima em diferentes processos industriais. / In the present estudy, ligninolytic fungi isolated from Brazilian ecosystems were evaluated for the ability to decolorize reactive dyes and a selected strain of Peniophora cinerea had its ligninolytic system characterized. Laccases from this fungi were evaluated for two biotechnology applications: decolorization of textile effluents and enzymatic hydrolysis of sugarcane bagasse. P. cinerea produced an halotolerant laccase activity, unique feature with the data available in the literature and with potential for biotechnological applications, making it the object of our studies. Laccase was the only ligninolytic enzyme detected in the extracellular extract from P. cinerea. The production of laccase by P. cinerea was evaluated in synthetic and complex media, in order to obtain higher levels of enzyme production. In complex medium (corn steep liquor 0.5%, sucrose 0.5% and copper, as inducer of laccase) P. cinerea produced about 1000 U/l of laccase. In synthetic medium containing tween 80 and xylidine, P. cinerea immobilized in polyurethane foam produced 3505 U/l of laccase in culture in flasks; in STR reactor P. cinerea produced only 150 U/l. Laccases produced by P. cinerea were purified and characterized. The supernatant of the culture medium was loaded on an anion exchange column, DEAE-Sepharose CL-6B, and three peaks with laccase activity were detected and applied separately on a Mono Q column. Eight isoenzymes of laccases with molecular weigh between 30 and 70 kDa and isoelectric point (pI) between 3 and 6 were revealed on electrophoresis gels at denaturing, native conditions and isoelectric foccusing (IEF). Some isoenzymes showed high thermostability at 50°C, optimal pH between 2.6 and 4.0, and Km for the ABTS between 12.7 and 20 ?M., depending on the type of laccase. Different mediators for laccases of P. cinerea were evaluated (HBT, HBA, 2,3 DHBA, DOPAC, ABTS, syringaldehyde, sodium oxalate and Mn2+). The system laccase, syringaldehyde, sodium oxalate and Mn2+ produced the highest decolorization of Reactive Blue 19. Laccases produced by P. cinerea were used for decolorization of a simulated efluent prepared with 100 mg/l of Reactive Red 271 hydrolyzed with NaOH 2 M and a real effluent, provided by a textile industry. The simulated efluent was completely decolorized within 24 hours by the enzymatic extract. In face of that, the real effluent was evaluated for decolorization by the extracts of P. cinerea and more than 60% of decolorization was obtained by laccase-mediator system. Laccases produced by P. cinerea were also used to hydrolize the sugar cane bagasse. Sugarcane bagasse samples were obtained by acid sulfite, alkaline sulfite, sulfuric acid, sodium chlorite or untreated (in natura). Two forms of treatment were evaluated: simultaneous treatment, ie, laccases have been added along with a commercial cellulase complex for hydrolysis of sugarcane bagasse, or a sequential treatment, in witch bagasse was pre-treated with laccase followed by cellulases. Simultaneous treatment with both enzymes resulted in lower conversion of cellulose than sequential treatment due to possible inactivation of cellulases by the oxidative action of laccase. Future studies of sequential treatment with laccase and hydrolysis of sugarcane bagasse should consider additional basic concepts of reactions such as: increase of enzyme load, temperature, reaction time and mediators. Laccases are exceptionally versatile for biotechnological applications and the production in large scale integrate the use of the enzyme in different industrial processes.
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Antifungal activity of essential oils associated with edible coatings for the postharvest control of spoilage fungi in strawberry (Fragaria spp.) / Atividade antifúngica de óleos essenciais associados a recobrimentos comestíveis para o controle pós-colheita de fungos deterioradores do morango (Fragaria spp.)Oliveira, Jacqueline de 05 February 2019 (has links)
Strawberry cultivation presents as limiting problem the phytosanitary management, which, associated with the residues of the toxic fungicides, the resistance of pathogens and the fragility of the fruit during transport and storage, make the commercialization of this product a challenge. The identification and development of new products with antifungal action, such as essential oils (EOs), is a promising alternative. Therefore, the present study addresses: the in vitro antifungal activity of three essential oils, Eucalyptus staigeriana, Lippia sidoides and Pimenta pseudocaryophyllus; the chemical composition and the mode of action of the EO with the highest potential and its in vivo activity when associated with carboxymethylcellulose (CMC), as well as its effects on the sensory quality and postharvest of strawberries. In chapters two, three and four, the studies were directed to Colletotrichum acutatum, Rhizopus stolonifer and Botrytis cinerea, respectively. In the last chapter, the antifungal activity of a commercial, alternative formulation with low toxicity was evaluated against the same three pathogens. Among the EOs evaluated, the one from L. sidoides presented the best antifungal activity, with the predominance of thymol in its composition, causing morphological alterations in the pathogens. in vivo, the strawberries treated with CMC associated to the EO presented a reduction in the severity of the diseases and improvements in the physicochemical properties with little alteration in the sensory quality of the fruit. The commercial formulation also presented a potential for the control, in vitro and in vivo, of the main postharvest strawberry-deteriorating fungi. / A cultura do morangueiro tem como problemática limitante o manejo fitossanitário, o que associado aos resíduos dos fungicidas tóxicos, à resistência dos patógenos e à fragilidade dos frutos durante o transporte e armazenamento, tornam a comercialização deste produto um desafio. A identificação e o desenvolvimento de novos produtos com ação antifúngica, como óleos essenciais (OEs), é uma alternativa promissora. Assim, o presente estudo discute: a atividade antifúngica in vitro de três óleos essenciais, Eucalyptus staigeriana, Lippia sidoides e Pimenta pseudocaryophyllus; a composição química e o modo de ação do OE de maior potencial e sua atividade in vivo quando associado com carboximetilcelulose (CMC), assim como seus efeitos na qualidade sensorial e pós-colheita dos morangos. Nos capítulos dois, três e quatro os estudos foram direcionados para Colletotrichum acutatum, Rhizopus stolonifer e Botrytis cinerea, respectivamente. No último capítulo avaliou-se a atividade antifúngica de uma formulação comercial, alternativa e de baixa toxidez contra os mesmos três patógenos. Dentre os OEs avaliados, o de L. sidoides apresentou melhor atividade antifúngica, com a predominância do timol em sua composição, causando alterações morfológicas nos patógenos. in vivo, os morangos tratados com CMC associado ao OE apresentaram redução na severidade das doenças e melhorias nas propriedades físico-químicas com pouca alteração na qualidade sensorial da fruta. A formulação comercial também apresentou potencial de controle, in vitro e in vivo, dos principais fungos deteriorantes pós-coheita do morango.
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Intensidade de míldio da videira em folha e podridão cinzenta em cacho em variedades viníferas sob diferentes sistemas de sustentação / Intensity of downy mildew in leaves and bunch rot in bunch on Vitis vinifera grapes in different training systemsBem, Betina Pereira de 13 August 2014 (has links)
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Previous issue date: 2014-08-13 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Santa Catarina highlands have been recognized for the production of fine wines with high quality. Downy mildew (Plamopara viticola) and bunch rot (Botrytis cinerea) are the most important diseases that occur in this winemaking region. The aim of this work was determinate the intensity of downy mildew in leaves and bunch rot in bunches under different training systems, as well the content of total polyphenols in skin of white varieties. Three experiments were done during 2012/2013 and 2013/2014 cycles, two of them in commercials vineyards with Cabernet Sauvignon, Sauvignon Blanc and Chardonnay in São Joaquim/SC subjected to Y-trellis and vertical shoot positioning trellis (VSP) and the third experiment in a experimental vineyard at Santa Catarina State University (CAV/UDESC) in Lages/SC with Merlot and Cabernet Sauvignon varieties, subjected to Simple Curtin, VSP, Geneva Double Curtin (GDC) and T-Trellis(TT) training systems. Based in the data obtained the temporal dynamic of downy mildew were quantified bi-weekly from the first symptoms appearance under natural conditions on leaves distributed in four medium-height branches on each five replications per treatment. The disease was compared by the beginning of symptoms appearance (BSA); time to reach the maximum
disease incidence and severity (TRMDI and TRMDS); maximum value of disease intensity and severity (Imax e Smax) and area under the disease progress curve (AUDPC) were calculated. The data were analyzed by linear regression and adjusted for the models Logistic, Monomolecular and Gompertz. The intensity of B. cinerea were evaluate at the harvest time, in five replicants where 30 randomily clusters were evaluated per treatment. The Y-trellis training system provided higher severity of downy mildew and bunch rot at Cabernet Sauvignon variety. The GDC system induced a high intensity of downy mildew at Merlot. Both white varieties evaluated, showed higher intensity of B. cinerea in Y-trellis when it was compared to VSP. The high concentration of total polyphenols was observed in Chardonnay subjected to Y-trellis system. The Gompertz model showed the best data fit. In conclusion, Y-trellis training system favors the development of downy mildew and bunch rot diseases when right management conditions are not adopted. VSP system showed the best disease control in this conditions and it is recommended to growing grapes to produce fine wines in highlands of Santa Catarina State / Regiões de elevada altitude de Santa Catarina (SC) vem se destacando pela produção de vinhos finos de alta qualidade. O míldio (Plamospara viticola) e a podridão cinzenta (Botrytis cinerea) da videira são as principais doenças que afetam o cultivo de uvas viníferas (Vitis vinifera) na região. O objetivo do trabalho foi avaliar a intensidade de míldio em folha e podridão cinzenta no cacho sob diferentes sistemas de sustentação, bem como o teor de polifenóis totais nas cascas de variedades brancas. Foram realizados três experimentos durante os ciclos 2012/2013 e 2013/2014, sendo dois ensaios em vinhedos comerciais em São Joaquim/SC nas variedades Cabernet Sauvignon, Chardonnay e Sauvignon Blanc sob os sistemas de sustentação espaldeira e ypsilon (Y) e o terceiro ensaio em vinhedo experimental da Universidade do Estado de Santa Catarina CAV/UDESC em Lages/SC nas variedades C. Sauvignon e Merlot sob sistemas de sustentação latada descontínua, Geneva Double Curtin (GDC), espaldeira e cortina simples. A dinâmica temporal do míldio foi avaliada quinzenalmente após o início do aparecimento dos sintomas, sob condições de infecção natural, em folhas distribuídas em 4 ramos medianos marcados aleatoriamente com 5 repetições por tratamento. A epidemia foi comparada em relação ao início do aparecimento dos sintomas (IAS), tempo para atingir a máxima
severidade e incidência da doença (TAMID e TAMSD), incidência máxima (Ymax), severidade máxima (Smax) e com os dados obtidos calculou-se as áreas abaixo da curva de progresso da incidência e severidade da doença (AACPID e AACPSD). Os dados foram analisados por regressão linear e ajustados para os modelos Logístico, Monomolecular e Gompertz. A intensidade de podridão cinzenta nos diferentes sistemas foi comparada no momento da colheita, em 5 repetições e 30 cachos aleatórios avaliados por tratamento. O sistema em ypsilon (Y) proporcionou uma maior intensidade de míldio e de podridão cinzenta da videira em relação ao sistema espaldeira, nas formas de manejo adotadas no vinhedo da
variedade C. Sauvignon. O sistema GDC apresentou maior intensidade de míldio para variedade Merlot. As variedades brancas apresentaram maior intensidade de B. cinerea no sistema em ypsilon (Y). O teor de polifenóis totais foi superior no sistema ypsilon (Y) para variedade Chardonnay. Dos modelos matemáticos testados o modelo Gompertz foi o modelo que permitiu melhor ajuste dos dados. O sistema em ypsilon (Y) favorece o desenvolvimento do míldio e da podridão cinzenta quando técnicas de manejo adequadas não são aplicadas. O sistema espaldeira mostrou maior controle fitossanitário nas condições adotadas, sendo recomendado para produção de uvas viníferas nas regiões de elevada altitude no Planalto Sul de Santa Catarina
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