Characterization of ligninolytic enzymes and metabolic profile of Cryphonectria parasitica and the isogenic converted strains by CHV1 hypovirus

Dupla diplomação com a Université Libre de TunisCryphonectria parasitica, the causal agent of Chestnut Blight, causes necrotic lesions (so-called cankers) on the bark of stems and branches of susceptible host trees. Cryphonectria Hypovirus 1 (CHV1) infects C. parasitica and reduces the fungus virulence (hypovirulence) and alters the fungus morphology in culture (pigmentation and sporulation capacity). By these characteristics the mycovirus CHV1 is used in Europe as a biological control agent of Chestnut Blight. The aim of this project is to better understand the effect of the mycovirus on the fungi pathogenicity by comparing the production of some lignin degrading enzymes and the metabolic profiles of some virulent and hypovirulent (converted and original) strains. For qualitative evaluation, several different compounds have been used as indicators for ligninolytic enzymes production. For quantitative evaluation, among nine strains five were chosen for biological tests and cultivation in minimal liquid media and the amount of enzyme produced were analyzed. Virulent strains were found to cause more damage in chestnut branches and to produce more lignin degrading enzymes. In apple fruits, some CHV1 infected strains produced bigger rot lesions than wild type strains. In parallel, Biolog FF MicroPlates have been used for the first time with C. parasitica strains to assess their metabolic profiles with concurrent reads of utilization of 95 different carbon sources. Moreover, carbohydrates, amino acids, amines/amides, miscellaneous and polymers were found to be more consumed by hypovirulent strains; therefore, this may suggest a novel adaptation mechanism in fungal ecology and fitness.Cryphonectria parasitica, o agente causal do cancro do castanheiro, provoca lesões necróticas (cancros) na casca do tronco e ramos de hospedeiros suscetíveis. O micovírus Cryphonectria hipovírus 1 (CHV1) infecta C. parasitica e reduz a virulência do fungo (hipovirulência) e altera a morfologia do fungo em cultura (capacidade de pigmentação e esporulação). Dadas essas características, o micovírus CHV1 é usado na Europa como agente de controlo biológico para o tratamento do cancro do castanheiro. O objetivo deste trabalho é entender melhor o efeito do micovírus na patogenicidade do fungo, comparando a produção de algumas enzimas que degradam a lenhina entre estirpes virulentas e hipovirulentas (convertidas e originais), assim como os perfis metabólicos. Para a avaliação qualitativa, vários compostos diferentes foram utilizados como indicadores para a produção de enzimas lenhinolíticas. Para a avaliação quantitativa, foram escolhidas cinco estirpes para testes biológicos e cultura em meio líquido mínimo, e a quantidade de enzima produzida analisada. Verificou-se que estirpes virulentas causam maior dimensão da lesãonos ramos do castanheiro e produzem mais enzimas lenhinolíticas. Em maçãs, algumas das estirpes hipovirulentas produziram lesões maiores do que as de tipo selvagem. Paralelamente, foram usadas pela primeira vez microplacas Biolog FF com estirpes de C. parasitica, para avaliar perfis metabólicos com leituras simultâneas de 95 fontes de carbono diferentes. Hidratos de carbono, aminoácidos, aminas / amidas, compostos diversos e polímeros foram mais consumidos pelas estirpes hipovirulentas; o que pode sugerir um mecanismo de adaptação ecológica do fungo

Virulencia, enzimas ligninolíticas y perfil metabólico de cryphonectria parasitica en cepas virulentas e hypovirulentas convertidas por CHV1 hipovirus

Cryphonectria parasitica, fungo responsável pelo cancro do castanheiro, causa lesões necróticas (cancros corticais) no tronco e ramos das árvores hospedeiras. O hipovírus, Cryphonectria hypovirus 1 (CHV1) reduz a virulência (hipovirulência) e altera a morfologia do fungo em cultura (redução da pigmentação e esporulação). As estirpes hipovirulentas CHV1 são utilizadas com sucesso na Europa como agentes de controlo biológico do Cancro do castanheiro. Objetivos: O objetivo deste trabalho foi compreender o efeito do hipovírus na virulência e metabolismo do fungo, comparando a produção de algumas enzimas lenhinolíticas e os perfis metabólicos de estirpes virulentas e estirpes isogénicas de C. parasitica convertidas (com CHV1). Métodos: A virulência de cada isolado foi avaliada por inoculação de micélio do fungo em maçãs (cv. Golden Delicious) e em ramos destacados de castanheiro com um ano de crescimento. Para a deteção da atividade de enzimas lenhinolíticas (lacases, peroxidases e celulases) foram usados vários substratos e compostos indicadores. O perfil metabólico de C. parasitica foi avaliado pelo sistema Biolog FF microplates pela utilização de 95 fontes diferentes de carbono. Resultados: A utilização de MicroPlacas FF (Biolog, Inc.) indicaram que a utilização de 95 fontes de carbono pelos cinco isolados de C. parasitica, foram significativamente diferentes (p <0,001), quando os substratos foram agrupados em seis tipos de compostos químicos. Os maiores valores de AWCD foram obtidos para os hidratos de carbono, ácidos carboxílicos e polímeros, e os menores valores para os grupos aminas / amidas, aminoácidos e compostos diversos. Conclusões: A avaliação da virulência de isolados de C. parasitica é importante para o estudo dos processos de hipovirulência mediados pelo hipovírus CHV1. Os ramos destacados de castanheiro foram, em nosso estudo, mais adequados que o teste em maçã para diferenciar as estirpes hipovirulentas das virulentas de C. parasitica. Os isolados virulentos evidenciaram sempre uma maior atividade de lacase induzida por ácido tânico (Lac3) e de outras enzimas lenhinolíticas (LiP, MnP e celulase) quando comparadas com os hipovirulentos. Os resultados da análise dos perfis metabólicos mostram que alguns grupos de substratos foram mais consumidos por isolados hipovirulentos. Estes estudos abrem novas perspetivas para entender o processo biológico usado pelo hipovírus, e sugerem que este é um método para discriminar estirpes hipovirulentas, e estudar a ecologia e a aptidão em campo destes isolados do fungo.Cryphonectria parasitica, the causal agent of chestnut blight, causes necrotic lesions (so-called cankers) on the bark of stems and branches of susceptible host trees. Cryphonectria hypovirus 1 (CHV1) infects C. parasitica and reduces the fungus virulence (hypovirulence) and alters the fungus morphology in culture (pigmentation and sporulation capacity). By these characteristics, the strains with hypovirus CHV1 are used in Europe as a biological control agent of chestnut blight. Objectives: The aim of this work is to understand the effect of hypovirus on fungi virulence by comparing the production of some lignin-degrading enzymes and the metabolic profiles of some isogenic virulent and hypovirulent (converted and original) strains. Methods: The virulence of each strain was evaluated by mycelial inoculation on apple fruits (cv. Golden Delicious) and on detached, one year old, chestnut branches. To detect the activity of ligninolytic enzymes (laccases, peroxidases and cellulases), various substrates and indicator compounds were used. The metabolic profile of C. parasitica was evaluated by the Biolog FF system using 95 different carbon sources. Results: Virulent strains were found to cause more significant necrotic lesions in chestnut branches (p<0.05) and to produce larger lignin-degrading enzymes. The use of Biolog FF MicroPlates indicated that the use of 95 carbon sources five isolates of C. parasitica were significantly different (p <0.001), when the substrates were grouped into six types of chemical compounds. The highest AWCD values were obtained for carbohydrates, carboxylic acids and polymers, and the lowest values for amines/amides, amino acids and miscellaneous. Conclusions: Virulence evaluation of C. parasitica strains is important to study the hypovirulence processes mediated by the hypovirus CHV1. The detached branches of chestnut were, in our study, more suitable than apple fruits test in discriminating hypovirulent from virulent strains of C. parasitica. Virulent strains showed higher activity of acid-tannic inducible laccase (Lac3) and other lignin-degrading enzymes (LiP, MnP, and cellulase) when compared with hypovirulent ones. The results of the metabolic profiles studies may lead to new perspectives for understanding the biological process used by the hypovirus therefore, this may suggest a method for discriminating hypovirulent strains and study ecology and field fitness in this fungal strains.Cryphonectria parasitica, el agente causal del Chancro del castaño, causa lesiones con necrosis (chancros corticales) en la corteza de los troncos y ramas de los árboles de hospedadores susceptibles. Cryphonectria hypovirus 1 (CHV1) y reduce la virulencia (hipovirulencia) con alteración de la morfología del hongo en cultivo (pigmentación y capacidad de esporulación). Por estas características, el micovirus CHV1 se usa en Europa como agente de control biológico del Chancro del castaño. Objetivos: El objetivo de este proyecto es comprender el efecto del hipovirus en la virulencia del hongo mediante la comparación de la producción de algunas enzimas ligninolíticas y de los perfiles metabólicos de algunas cepas virulentas e hipovirulentas (convertidas y originales). Métodos: La virulencia de cada aislado se evaluó inoculando el micelio del hongo en manzanas (cv. Golden Delicious) y en ramas separadas de castaño con un año de crecimiento. Para la detección de la actividad de las enzimas ligninolíticas (lacasas, peroxidasas y celulasas) se utilizaron varios sustratos y compuestos indicadores. El perfil metabólico de C. parasitica fue evaluado por el sistema Biolog FF utilizando 95 fuentes de carbono distintas. Resultados: Se constató que las cepas virulentas causan lesiones significativamente más grandes en las ramas de castaño (p < 0,05) y producen más enzimas ligninolíticas. El uso de Biolog FF MicroPlates indicaron que el uso de 95 fuentes de carbono por los cinco aislados de C. parasitica eran significativamente diferentes (p <0.001), cuando los sustratos se agruparon en seis tipos de compuestos químicos. Los valores más altos de AWCD se obtuvieron para carbohidratos, ácidos carboxílicos y polímeros, y los valores más bajos para las aminas /amidas, aminoácidos y compuestos diversos. Conclusiones: La evaluación de la virulencia de las cepas de C. parasitica es importante para estudiar los procesos de hipovirulencia mediados por el hipovirus CHV1. Las ramas de castaño fueron, en nuestro estudio, más adecuadas que las manzanas para discriminar cepas hipovirulentas y virulentas de C. parasitica. Los aislados virulentos mostraron una mayor actividad de lacasa inducible por ácido tánico (Lac3) y otras enzimas ligninolíticas (LiP, MnP y celulasa) en comparación con los hipovirulentos. Los resultados de los perfiles metabólicos obtenidos muestran que algunos grupos químicos de sustratos fueron más consumidos por cepas hipovirulentas. Estos estudios pueden conducir a nuevas perspectivas para entender el proceso biológico utilizado por el hipovirus, por lo tanto, esto puede sugerir que este es un método para discriminar cepas hipovirlulentas y para estudiar la ecología y la aptitud de campo de estas cepas de hongos.This work supported by the project PDR2020-101-030959 - “BioChestnut- IPM - Implementar estratégias de luta eficazes contra doenças do castanheiro e amendoeira”.info:eu-repo/semantics/publishedVersio

Virulence, ligninolytic enzymes and metabolic profile of Cryphonectria parasitica virulent and hypovirulent strains Converted by CHV1 hypovirus

Introduction: Cryphonectria parasitica, the causal agent of chestnut blight, causes necrotic lesions (so-called cankers) on the bark of stems and branches of susceptible host trees. Cryphonectria hypovirus 1 (CHV1) infects C. parasitica and reduces the fungus virulence (hypovirulence) and alters the fungus morphology in culture (pigmentation and sporulation capacity). By these characteristics, the strains with hypovirus CHV1 are used in Europe as a biological control agent of chestnut blight. Objectives: The aim of this work is to understand the effect of hypovirus on fungi virulence by comparing the production of some lignin-degrading enzymes and the metabolic profiles of some isogenic virulent and hypovirulent (converted and original) strains. Methods: The virulence of each strain was evaluated by mycelial inoculation on apple fruits (cv. Golden Delicious) and on detached, one year old, chestnut branches. To detect the activity of ligninolytic enzymes (laccases, peroxidases and cellulases), various substrates and indicator compounds were used. The metabolic profile of C. parasitica was evaluated by the Biolog FF system using 95 different carbon sources. Results: Virulent strains were found to cause more significant necrotic lesions in chestnut branches (p<0.05) and to produce larger lignin-degrading enzymes. The use of Biolog FF MicroPlates indicated that the use of 95 carbon sources five isolates of C. parasitica were significantly different (p <0.001), when the substrates were grouped into six types of chemical compounds. The highest AWCD values were obtained for carbohydrates, carboxylic acids and polymers, and the lowest values for amines/amides, amino acids and miscellaneous. Conclusions: Virulence evaluation of C. parasitica strains is important to study the hypovirulence processes mediated by the hypovirus CHV1. The detached branches of chestnut were, in our study, more suitable than apple fruits test in discriminating hypovirulent from virulent strains of C. parasitica. Virulent strains showed higher activity of acid-tannic inducible laccase (Lac3) and other lignin-degrading enzymes (LiP, MnP, and cellulase) when compared with hypovirulent ones. The results of the metabolic profiles studies may lead to new perspectives for understanding the biological process used by the hypovirus therefore, this may suggest a method for discriminating hypovirulent strains and study ecology and field fitness in this fungal strains.Introducción: Cryphonectria parasitica, el agente causal del Chancro del castaño, causa lesiones con necrosis (chancros corticales) en la corteza de los troncos y ramas de los árboles de hospedadores susceptibles. Cryphonectria hypovirus 1 (CHV1) y reduce la virulencia (hipovirulencia) con alteración de la morfología del hongo en cultivo (pigmentación y capacidad de esporulación). Por estas características, el micovirus CHV1 se usa en Europa como agente de control biológico del Chancro del castaño. Objetivos: El objetivo de este proyecto es comprender el efecto del hipovirus en la virulencia del hongo mediante la comparación de la producción de algunas enzimas ligninolíticas y de los perfiles metabólicos de algunas cepas virulentas e hipovirulentas (convertidas y originales). Métodos: La virulencia de cada aislado se evaluó inoculando el micelio del hongo en manzanas (cv. Golden Delicious) y en ramas separadas de castaño con un año de crecimiento. Para la detección de la actividad de las enzimas ligninolíticas (lacasas, peroxidasas y celulasas) se utilizaron varios sustratos y compuestos indicadores. El perfil metabólico de C. parasitica fue evaluado por el sistema Biolog FF utilizando 95 fuentes de carbono distintas. Resultados: Se constató que las cepas virulentas causan lesiones significativamente más grandes en las ramas de castaño (p < 0,05) y producen más enzimas ligninolíticas. El uso de Biolog FF MicroPlates indicaron que el uso de 95 fuentes de carbono por los cinco aislados de C. parasitica eran significativamente diferentes (p <0.001), cuando los sustratos se agruparon en seis tipos de compuestos químicos. Los valores más altos de AWCD se obtuvieron para carbohidratos, ácidos carboxílicos y polímeros, y los valores más bajos para las aminas /amidas, aminoácidos y compuestos diversos. Conclusiones: La evaluación de la virulencia de las cepas de C. parasitica es importante para estudiar los procesos de hipovirulencia mediados por el hipovirus CHV1. Las ramas de castaño fueron, en nuestro estudio, más adecuadas que las manzanas para discriminar cepas hipovirulentas y virulentas de C. parasitica. Los aislados virulentos mostraron una mayor actividad de lacasa inducible por ácido tánico (Lac3) y otras enzimas ligninolíticas (LiP, MnP y celulasa) en comparación con los hipovirulentos. Los resultados de los perfiles metabólicos obtenidos muestran que algunos grupos químicos de sustratos fueron más consumidos por cepas hipovirulentas. Estos estudios pueden conducir a nuevas perspectivas para entender el proceso biológico utilizado por el hipovirus, por lo tanto, esto puede sugerir que este es un método para discriminar cepas hipovirlulentas y para estudiar la ecología y la aptitud de campo de estas cepas de hongos

Virulence, ligninolytic enzymes and metabolic profile of Cryphonectria parasitica virulent and hypovirulent strains Converted by CHV1 hypovirus

Introduction: Cryphonectria parasitica, the causal agent of chestnut blight, causes necrotic lesions (so-called cankers) on the bark of stems and branches of susceptible host trees. Cryphonectria hypovirus 1 (CHV1) infects C. parasitica and reduces the fungus virulence (hypovirulence) and alters the fungus morphology in culture (pigmentation and sporulation capacity). By these characteristics, the strains with hypovirus CHV1 are used in Europe as a biological control agent of chestnut blight. Objectives: The aim of this work is to understand the effect of hypovirus on fungi virulence by comparing the production of some lignin-degrading enzymes and the metabolic profiles of some isogenic virulent and hypovirulent (converted and original) strains. Methods: The virulence of each strain was evaluated by mycelial inoculation on apple fruits (cv. Golden Delicious) and on detached, one year old, chestnut branches. To detect the activity of ligninolytic enzymes (laccases, peroxidases and cellulases), various substrates and indicator compounds were used. The metabolic profile of C. parasitica was evaluated by the Biolog FF system using 95 different carbon sources. Results: Virulent strains were found to cause more significant necrotic lesions in chestnut branches (p<0.05) and to produce larger lignin-degrading enzymes. The use of Biolog FF MicroPlates indicated that the use of 95 carbon sources five isolates of C. parasitica were significantly different (p <0.001), when the substrates were grouped into six types of chemical compounds. The highest AWCD values were obtained for carbohydrates, carboxylic acids and polymers, and the lowest values for amines/amides, amino acids and miscellaneous. Conclusions: Virulence evaluation of C. parasitica strains is important to study the hypovirulence processes mediated by the hypovirus CHV1. The detached branches of chestnut were, in our study, more suitable than apple fruits test in discriminating hypovirulent from virulent strains of C. parasitica. Virulent strains showed higher activity of acid-tannic inducible laccase (Lac3) and other lignin-degrading enzymes (LiP, MnP, and cellulase) when compared with hypovirulent ones. The results of the metabolic profiles studies may lead to new perspectives for understanding the biological process used by the hypovirus therefore, this may suggest a method for discriminating hypovirulent strains and study ecology and field fitness in this fungal strains.info:eu-repo/semantics/publishedVersio