Biocomposites de base quitosano para la protección de especies agricolas, forestales y de madera

PROGRAMA DE DOCTORADO EN CIENCIA E INGENIERÍA AGROALIMENTARIA Y DE BIOSISTEMAS TESIS DOCTORAL: Biocomposites antifúngicos de base quitosano para la protección de especies agrícolas, forestales y de madera Presentada por Iosody Silva Castro para optar al grado de Doctora por la Universidad de Valladolid Dirigida por: Dr. Jesús Martín Gil Dr. Miguel Ángel Méndez Rojas No quiero convencer a nadie de nada… Tratar de convencer a otra persona es indecoroso, es atentar contra su libertad de pensar o de creer o de hacer lo que le dé la gana. Yo quiero solo enseñar, dar a conocer, mostrar, no demostrar. Que cada uno llegue a la verdad por sus propios pasos, y que nadie le llame equivocado o limitado. (¿Quién es quién para decir esto es así, si la historia de la humanidad no es más que una historia de contradicciones y de tanteos y de búsquedas?) Si a alguien he de convencer algún día, ese alguien he de ser yo mismo. Convencerme de que no vale la pena llorar, ni afligirse, ni pensar en la muerte. “La vejez, la enfermedad y la muerte”, de Buda, no son más que la muerte, y la muerte es inevitable. Tan inevitable como el nacimiento. Lo bueno es vivir del mejor modo posible. Peleando, lastimando, acariciando, soñando. (¡pero siempre se vive del mejor modo posible!) Mientras yo no pueda respirar bajo el agua, o volar (pero de verdad volar, yo solo, con mis brazos), tendrá que gustarme caminar sobre la tierra, y ser hombre, no pez ni ave. No tengo ningún deseo que me digan que la luna es diferente a mis sueños. Jaime Sabines Poeta Mexicano Agradecimientos ¿Qué tienen que ver una gamba, una abeja y la cubertería de plata de la abuela? En esta Tesis Doctoral lo cuento en más de 3 minutos, de hecho lo cuento profundamente, de la misma forma en que agradezco a todos; todos los que de múltiples formas aportaron sus granitos de arena y/o cáscaras de gambas para el desarrollo de este proyecto de investigación. En primer lugar agradezco a mis directores, Jesús Martín Gil y Miguel Ángel Méndez Rojas, por su apoyo ilimitado en cada etapa del desarrollo de mi doctorado, en la investigación en el laboratorio, la publicación de los resultados, las estancias en el extranjero y la revisión de la Tesis, por nombrar algunas. Al Consejo Nacional de Ciencia y Tecnología de México (CONACYT), demás está decir que su apoyo ha sido fundamental, gracias por la beca de estudios en el extranjero que me ha sido concedida. A los directores de los laboratorios en los que tuve el honor de trabajar durante las estancias en la Universidad Federal de Viçosa, Brasil, el Dr. Sergio Motoike, la Dra. Marisa Vieira y el Dr. Robert Barreto; y en la Universidad de las Américas Puebla, México, el Dr. José Luis Sánchez Salas. Siempre es necesario más tiempo del previsto, sin embargo, aprendí mucho más de que lo había podido imaginar gracias a su enorme experiencia. Mi más sincero agradecimiento a todos los profesores de la ETSIIAA que desde el primer momento aceptaron colaborar conmigo en esta investigación y que han estado al pendiente hasta el punto final de la Tesis, Luis Acuña, Milagros Casado, Jorge Martín García, Julio Diez, Agustín León, Salvador Hernández, Luis Manuel Navas y Pablo Martín Ramos. A mis compañeras/amigas de laboratorio, de despacho y de piso, Mihaela, Amparo, Marciabela, Sabina, Carmen, Priscila, Lupita, Laura… Y por supuesto, con todo el cariño del mundo agradezco a mi familia entera, mi fe, mi impulso, mi fuerza, el hilo que mantuvo atado mi corazón a México; a mi mamá, mi papá, a Nain, Yohalit y Marcelino (mis hermanos), a mis abuelos, tíos, primos… A todas las personas maravillosas con las que he coincidido estos años y que desde entonces son mis amigos, en especial al mejor de todos… Biocomposites antifúngicos de base quitosano para la protección de especies agrícolas, forestales y de madera | 1 Contenido Resumen ................................................................................................................................. 2 Abstract .................................................................................................................................. 3 1. INTRODUCCIÓN GENERAL .............................................................................................. 4 1.1 Quitosano, un polímero natural .............................................................................. 4 1.2 Propóleo y nanoplata, otros materiales antifúngicos ............................................. 9 1.3 Composites de base quitosano .............................................................................. 12 1.4 Composites de quitosano para la sanidad agroforestal ........................................ 18 2. Marco de referencia y estructura de la tesis ................................................................ 22 3. OBJETIVOS .................................................................................................................... 25 4. MATERIALES Y MÉTODOS ............................................................................................. 27 4.1 Reactivos, material biológico, equipos y softwares .............................................. 27 4.2 Síntesis de composites ........................................................................................... 28 4.3 Pruebas in vitro ...................................................................................................... 29 4.4 Pruebas in vivo ....................................................................................................... 29 4.5 Análisis estadístico ................................................................................................. 29 5. RESULTADOS PRINCIPALES ........................................................................................... 30 5.1 Análisis molecular, síntesis y caracterización de composites ................................ 30 5.2 MIC y efectividad in vitro ....................................................................................... 31 5.3 Control de enfermedades de especies agrícolas, forestales y madera ................. 32 6. DISCUSIÓN GENERAL .................................................................................................... 34 7. CONCLUSIONES ............................................................................................................. 38 7.1 Conclusions ............................................................................................................ 40 8. Perspectivas de la investigación ................................................................................... 42 Referencias ........................................................................................................................... 44 Artículos originales ............................................................................................................... 53 Biocomposites antifúngicos de base quitosano para la protección de especies agrícolas, forestales y de madera | 2 Resumen El quitosano es uno de los polímeros de origen natural más interesantes de las últimas décadas. Es la forma desacetilada de la quitina, el segundo polisacárido natural más abundante en el planeta. Su actividad antimicrobiana, además de su naturaleza biodegradable, biocompatible y no tóxica le ha conferido un extenso número de aplicaciones en distintas áreas, incluida la agricultura. La versatilidad de su estructura, así como sus propiedades de sorción de macromoléculas y quelación de partículas metálicas, gracias a su carácter catiónico derivado del grupo amino, le otorgan un mayor interés y la facilidad de generar nuevos materiales que potencien su actividad. No obstante, las extensas aportaciones de la investigación sobre los productos derivados de quitosano, en sus formas, síntesis, aplicaciones, incluso a nivel teórico en la descripción de su comportamiento químico y de los mecanismos de acción que ejercen, no cesan, por el contrario su desarrollo continúa en auge. En la presente tesis doctoral se realiza en primer lugar una revisión del estado del arte de la síntesis de nuevos compuestos basados en quitosano, que incluyen desde extractos naturales hasta nanopartículas metálicas, mediante la utilización de nuevas tecnologías. Se revisan también sus aplicaciones como antimicrobiano para diversos materiales que deban mantenerse y conservarse de microorganismos y patógenos. Posteriormente, se realiza por primera vez un análisis molecular de la N-acetil-D-glucosamina (la molécula constituyente de la quitina y el quitosano) por combinación de técnicas de espectroscopía de THz-TDS y FTIR con estudios teóricos basados en métodos semiempíricos de química cuántica. Se propuso el diseño y la síntesis de un nuevo material compuesto a partir de oligómeros de quitosano, óxido de grafeno reducido y nanopartículas de plata, mediante una técnica asistida por microondas; consiguiendo un material funcionalizado y estable, como evidencia la caracterización realizada por UV-Vis, FTIR, XRD y TEM. Se prepararon también nuevos materiales compuestos a partir de quitosano, oligómeros de quitosano, propóleo y nanoplata. Estos composites fueron caracterizados por FTIR y se llevaron a cabo estudios in vitro e in vivo para evaluar su efectividad contra hongos fitopatógenos de interés agrícola, forestal y de la industria de la madera. Se consiguió controlar con éxito hongos devastadores como Hemileia vastatrix causante de grandes pérdidas económicas por la enfermedad de la roya de las hojas del café, Fusarium circinatum causante del chancro resinoso de los pinos, el hongo más dañino de bosques de coníferas a nivel mundial y Trametes versicolor causante de la pudrición blanca de la madera, entre otros. Los análisis in vivo han demostrado la potencialidad del quitosano y productos derivados como una herramienta -más respetuosa con el medio ambienteideal para ser incluida en los programas de manejo integrado para el control de las enfermedades el ámbito agroforestal. Palabras Clave: copolímeros, oligómeros de quitosano, propóleo, nanoplata, fitopatógenos Biocomposites antifúngicos de base quitosano para la protección de especies agrícolas, forestales y de madera | 3 Abstract Chitosan is one of the most interesting natural polymers of recent decades. It is the acetylated form of chitin, the second most abundant natural polysaccharide on the planet. Its antimicrobial activity, in addition to its biodegradable, biocompatible and non-toxicity nature, has conferred an extensive number of applications in different areas, including agriculture. The versatility of its structure, as well as its sorption properties of macromolecules and chelation of metallic particles, due to its cationic character derived from the amino group, give it a greater interest and the ease of generating new materials that enhance its activity. However, the extensive contributions of research on chitosanderived products, in their form, synthesis, applications, even at the theoretical level in the description of their chemical behavior and the mechanisms of action they exert, do not stop, on the contrary its development continues to rise. In the present doctoral thesis, a review of the state of the art of the synthesis of new compounds based on chitosan, ranging from natural extracts to metal nanoparticles, is made through the use of new technologies. Its applications as an antimicrobial are also reviewed for various materials that must be maintained and conserved from microorganisms and pathogens. Subsequently, a molecular analysis of N-acetyl-D-glucosamine (the constituent molecule of chitin and chitosan) is performed for the first time by combining THz-TDS and FTIR spectroscopy techniques with theoretical studies based on semiempirical quantum chemistry methods. The design and synthesis of a new composite material was proposed from chitosan oligomers, reduced graphene oxide and silver nanoparticles, by means of a microwave-assisted technique; obtaining a functionalized and stable material, as evidenced by the characterization made by UV-Vis, FTIR, XRD and TEM. New composite materials were also prepared from chitosan, chitosan oligomers, propolis and nanosilver. These composites were characterized by FTIR and in vitro and in vivo studies were carried out to evaluate their effectiveness against phytopathogenic fungi of agricultural, forest and wood industry species. Achieving successful control of devastating fungi such as Hemileia vastatrix, which causes coffee leaf rust (CLR), the worst disease of coffee worldwide; Fusarium circinatum, that causes pine pitch canker (PPC), the most harmful fungus of coniferous forests worldwide; and Trametes versicolor which causes white rot decay of the wood, among others. The in vivo analyzes have demonstrated the potential of chitosan and derived products as a tool -more environmentally friendlyideal to be included in an integrated management approach for the control of diseases in the agriculture and forestry fields. Keywords: copolymers, chitosan oligomers, propolis, nanosilver, phytopathogens Biocomposites antifúngicos de base quitosano para la protección de especies agrícolas, forestales y de madera | 4 1. INTRODUCCIÓN GENERAL 1.1 Quitosano, un polímero natural El quitosano es un polisacárido lineal compuesto por cadenas de N-glucosamina, se deriva de la deacetilización de la quitina, el segundo polímero natural más abundante en el planeta, después de la celulosa (Shahidi et al. 1999) y con una gran similitud entre sí. La quitina es el componente estructural de las paredes celulares de hongos, del exoesqueleto de artrópodos y de órganos de otros organismos invertebrados (Se-Kwon Kim 2011; Zakrzewski et al. 2014). El quitosano y sus derivados han sido de los polímeros de origen natural de mayor interés en las últimas décadas, especialmente por su actividad antimicrobiana y sus características de biocompatibilidad, no toxicidad y biodegradabilidad (Ma et al. 2017). 1.1.1 Origen La quitina, fue descrita por primera vez por Henri Braconnot en 1811, quien logró aislarla de hongos superiores llamándole fungina. Posteriormente, Auguste Odier en 1823 descubrió una sustancia similar al tejido de las plantas en la cutícula de los insectos, llamándola “quitina” del griego χιτών (túnica, envoltura). El quitosano, por su parte, fue descubierto en 1859 por Rouget, cuando trató quitina -insoluble en aguacon una solución muy concentrada de hidróxido de potasio a altas temperaturas y esta se disolvió en ácidos orgánicos. No obstante, fue hasta 1894 que Hoppe-Seyler le llamó quitosano y hasta 1950, que fue claramente descrito como un polímero compuesto por glucosamina, del mismo modo que la quitina por N-acetilglucosamina. A partir de ese año el interés por su uso en aplicaciones químicas e industriales, creció rápidamente (Muzzarelli et al. 2012). El quitosano también puede encontrarse naturalmente en algunos organismos, sin embargo, su fuente de producción a escala industrial deriva de la N-desacetilación termoalcalina de quitina aislada de deshechos de crustáceos (Goycoolea et al. 2009). Aunque se ha estimado que 1×1010 t de quitina se encuentran constantemente en la biósfera (Hamed et al. 2016), las dos especies principales de crustáceos marinos aprovechadas como fuente de quitina para la producción de quitosano son los camarones y cangrejos (Rinaudo 2006). El exoesqueleto de estos invertebrados consiste en un tejido fibroso mineralizado a base de quitina, con diferentes niveles estructurales (Figura 1). A nivel molecular, presenta una alineación antiparalela formando cristales de α-quitina (Fig 1a), que se encuentran en una disposición de 18 a 25 moléculas en forma de unidades cristalinas estrechas y largas (Fig 1b), envueltas por proteínas que forman nanofibras de 2 a 5 nm de diámetro y 300 nm de longitud (Fig 1c). Estas nanofibras se agrupan en fibras más largas de quitina-proteína, de | Introducción General Biocomposites antifúngicos de base quitosano para la protección de especies agrícolas, forestales y de madera | 5 50 a 300 nm de diámetro (Fig 1d), formando una red plana tejida y periódicamente ramificada (Fig 1e). El espacio entre las fibras se rellena con proteínas y biominerales de tamaño nanoscópico y microscópico, principalmente CaCO3 cristalino, aunque también pueden aparecer partículas amorfas según la especie de crustáceo. En el siguiente nivel, se forma una capa por el apilamiento helicoidal de las capas de proteína en planos que giran gradualmente alrededor de su eje (Fig 1f), creando así complejas estructuras como la endocutícula, la exocutícula y la epicutícula (Fig 1g), las cuales se caracterizan por ser fuertes estructuras mineralizadas, diferenciadas jerárquicamente de menor a mayor rigidez (Raabe et al. 2005). Figura 1. Niveles estructurales de la composición del exoesqueleto de los crustáceos formado a base de quitina (ejemplo: Homarus americanus). 1.1.2 Estructura química El quitosano, producto de la quitina desacetilada, presenta una estructura química similar, parecida también a la celulosa (Figura 2). La quitina a diferencia de la celulosa, posee un grupo acetilamida (-NHCOCH3) enlazado al carbono 2 (C-2) del anillo de piranosa. El quitosano, por su parte, difiere de la quitina por la ausencia del grupo acetil, de tal forma que en el C-2 contiene un grupo amino (NH2). Sin embargo, el quitosano en realidad está compuesto de cadenas lineales distribuidas aleatoriamente de β-(1-4)-2-amino-2-desoxi-Dglucopiranosa y β-(1-4)-2-acetamido-2-desoxi-D-glucopiranosa, unidades desacetiladas y acetiladas, respectivamente, en proporciones variantes a partir de 80:20 según el grado de pureza o desacetilación (Zou et al. 2016). La presencia del NH2 le confiere un carácter catiónico, siendo el único polímero natural con esta característica (Kean & Thanou 2010). El grupo amino en el quitosano tiene un valor de pKa de 6,5 (Thakur & Thakur 2014); por lo que es un polímero sensible a los valores del pH; | Introducción General Biocomposites antifúngicos de base quitosano para la protección de especies agrícolas, forestales y de madera | 12 Figura 7. Micrografía electrónica de nanopartículas de plata en una matriz de quitosano. 1.3 Composites de base quitosano En la creciente importancia del uso de biopolímeros, el quitosano ha sido uno de los más utilizado en las últimas décadas para la formación de materiales compuestos. Como se mencionó anteriormente, una de la grandes ventajas del quitosano son la capacidad de sorción y quelación, conferidas por el grupo amino y la funcionalidad del alcohol primario (OH) (Velmurugan et al. 2009), generados en la desacetilación de la quitina y en la degradación de la cadena polimérica para la generación de oligómeros. La formación de los nuevos enlaces no se limita a un grupo de especies químicas, pues se han reportado composites de quitosano con macromoléculas orgánicas como con nanopartículas metálicas (Saharan et al. 2015; Vieira et al. 2016). Las técnicas de síntesis desde la producción de quitosano a partir de quitina, la formación de oligómeros y la composición de nuevos materiales involucran diversos procesos físicos, químicos y biológicos. Por otra parte, la caracterización fisicoquímica de estos nuevos materiales es muy importante, no solo como control de los procesos de síntesis, también para evaluar su actividad y sus potenciales aplicaciones. 1.3.1 Técnicas de síntesis De quitina a quitosano Las fuentes de quitina para la producción comercial de quitosano son principalmente las cáscaras de crustáceos marinos. Una vez procesados estos residuos, mediante el lavado y la molienda de los mismos, el siguiente paso es someterlos a un proceso químico que consiste en 3 pasos fundamentales: desmineralización ácida, desproteinización alcalina y desacetilación alcalina como se representa en la Figura 8 (Leceta et al. 2013). | Introducción General Biocomposites antifúngicos de base quitosano para la protección de especies agrícolas, forestales y de madera | 13 Figura 8. Proceso esquemático de la producción de quitosano El primer paso, la disolución de los minerales, se realiza con ácido clorhídrico (HCl) concentrado (1N) para disolver el carbonato cálcico (CaCO3) presente de 30 a 50% en la composición de estos residuos. El segundo paso, la degradación de las proteínas, se lleva a cabo con hidróxido de sodio (NaOH) diluido; pues del 30 al 40% del exoesqueleto de los crustáceos está compuesto por proteínas. El resultado de este segundo paso es la quitina aislada, generalmente α-quitina (más abundante). Después puede realizarse una decoloración con hipoclorito de sodio (NaOCl) diluido, para remover pigmentos de carotenoides como la astaxantina, que se encuentran en el 20 o 30% de peso seco de los residuos. El último paso es la desacetilación de la quitina; esta es la remoción de los grupos acetilos (COCH3) y puede llevarse a cabo a través de un tratamiento térmico acompañado de un proceso químico de hidrólisis alcalina con NaOH concentrado o biológico vía enzimática con algunos hongos y bacterias, durante tiempos prolongados de reacción. Posteriormente se realiza un lavado y secado del material, como resultado obtenemos partículas sólidas de quitosano (Aranaz et al. 2009; Pillai et al. 2011; Benhabiles et al. 2012). De quitosano a oligómeros La aplicación de los oligómeros de quitosano presenta mayor interés debido a que mejoran algunas desventajas del quitosano con más alto GP, como su alta viscosidad y baja solubilidad en agua (Benhabiles et al. 2012; Zou et al. 2016). Las técnicas de degradación de las cadenas poliméricas consisten en el rompimiento de los enlaces glucósidos β-(1-4) entre cada molécula de glucosamina (como se mostró en la figura 3), para generar cadenas más pequeñas u oligómeros de aproximadamente 2 a 10 monómeros (Verlee et al. 2017). Algunos de los métodos físicoquímicos y biológicos más utilizados en la despolimerización del quitosano son la degradación oxidativa (Sun et al. 2007; Xia et al. 2013), hidrólisis ácida Cáscaras de crustáceos Quitosano Desmineralización Desproteinización Desacetilación - HCl - NaOH - NaOH Quitina | Introducción General Biocomposites antifúngicos de base quitosano para la protección de especies agrícolas, forestales y de madera | 14 (Tsao et al. 2011), hidrólisis enzimática (Kim & Rajapakse 2005), irradiación (Duy et al. 2011) y ultrasonidos (Savitri et al. 2014), entre otras donde se combinan dos o más de estas reacciones. El mecanismo de degradación oxidativa, puede llevarse a cabo con peróxido de hidrógeno (H2O2) como material oxidante, adicionándolo a una solución de quitosano diluido en medio ácido (ácido acético, láctico, etc.) para que una vez distribuidas las cadenas poliméricas en el medio, entren en contacto con los radicales libres formados por la disociación del H2O2, estos tienen la capacidad de atacar los enlaces glucosídicos de los polisacáridos (Sun et al. 2007). Composites orgánicos e inorgánicos Los métodos de síntesis para generar materiales compuestos entre moléculas de quitosano y macromoléculas orgánicas se basan en la capacidad de sorción de este polímero. El mecanismo de sorción, puede establecerse a través de distintas vías: la interacción con el N de la amina, con el O del hidroxilo o ambos (Verlee et al. 2017). Tal es el caso del material compuesto por quitosano-propóleo, se ha reportado que los polifenoles del propóleo pueden formar enlaces de hidrógeno y enlaces covalentes con los grupos funcionales de la glucosamina del quitosano o sus oligómeros (Siripatrawan & Vitchayakitti 2016). Una de las ventajas de esta unión es que ambos compuestos presentan un mayor poder antimicrobiano respecto a la aplicación de los mismos individualmente (Torlak & Sert 2013; Rollini et al. 2017). Por otra parte, los mecanismos de síntesis para la producción y estabilización de nanopartículas metálicas, se basan en la propiedad de quelación de iones metálicos del quitosano. Se sabe que los grupos amino tienden a formar complejos, por ejemplo, con los iones Ag+ de la nanoplata, con los que forma un enlace coordinado (Wei et al. 2009). Además de una síntesis efectiva de las nanopartículas con el uso del quitosano como reductor y la estabilización de las mismas por la quelación, se han encontrado mejores propiedades antibacterianas y antifúngicas con el uso de este compuesto (Saharan et al. 2013; Chowdappa et al. 2014; Wang et al. 2015). También existe el caso donde se requieren ambos mecanismo de síntesis, para producir composites ternarios, entre quitosano, especies orgánicas y especies inorgánicas. Por ejemplo, en un material de oligómeros de quitosano, con óxido de grafeno reducido (rGO) y nanopartículas de plata, los oligómeros se unen al óxido de grafeno a través de la sorción, creando un copolímero. Posteriormente, las nanopartículas de plata se enlazan a la matriz polimérica, principalmente a las hojas del rGO que brindan mayor área superficial para la deposición de las nanopartículas, que junto a los oligómeros se obtiene un material estable y funcionalizado para diversas aplicaciones (Marta et al. 2015). Entre otros como se muestran en la tabla 1. | Introducción General Biocomposites antifúngicos de base quitosano para la protección de especies agrícolas, forestales y de madera | 15 Tabla 1. Materiales compuestos de base quitosano, con macromoléculas orgánicas y nanopartículas metálicas. Naturaleza del material Composite Síntesis Aplicación Referencia Macromoléculas orgánicas Quitosano–almidón Método de fundición Recubrimiento antifúngico de papayas Escamilla-García et al. 2018 Quitosano–Aloe vera Método de fundición Recubrimiento antifúngico de moras Vieira et al. 2016 Quitosano– cinamaldehido Enlace por base de Shiff Empaque antifúngico de alimentos Demitri et al. 2016 Quitosano–saponina Gelificación iónica Protección antifúngico de cultivos Saharan et al. 2013 Quitosano–aceites esenciales Método de fundición Empaque antifúngico de alimentos Avila-Sosa et al. 2012 Nanopartículas metálicas Quitosano– nanocobre Gelificación iónica Inductor de crecimiento y antifúngico en tomate Saharan et al. 2015 Quitosano– nanoplata Un solo paso – tratamiento químico Antifúngico Wang et al. 2015 Quitosano– nanoplata Un solo paso – tratamiento térmico Antifúngico – contra antracnosis del mango Chowdappa et al. 2014 Quitosano– nanocobre Gelificación iónica Antifúngico - Protección de cultivos Saharan et al. 2013 Ambos (orgánico e inorgánico) Quitosano–aceite de neem– nanopartículas de óxido de zinc Método de fundición Antibacteriano – empaques de alimentos Sanuja et al. 2015 1.3.2 Técnicas de caracterización El desarrollo de nuevos materiales involucra el desarrollo de nuevas técnicas, o por lo menos, de técnicas más sensibles para su análisis. Entre las técnicas más utilizadas para el estudio y caracterización de polímeros como el quitosano, sus derivados y materiales compuestos basados a partir de su estructura tenemos la espectroscopía y la microscopía, como dos grandes áreas con muchas herramientas disponibles, entre otras técnicas características destacadas en la tabla 2. | Introducción General Biocomposites antifúngicos de base quitosano para la protección de especies agrícolas, forestales y de madera | 16 Técnicas espectroscópicas UV-vis. La espectroscopía o espectrofotometría UV-visible, consiste en la emisión de fotones, o radiación electromagnética, en las regiones del espectro visible, ultravioleta cercano (UV) e infrarrojo cercano (IR), por tanto abarca longitudes de onda desde 380 nm a 780 nm. A través de esta técnica es posible cuantificar las transiciones electrónicas en las moléculas causadas por la absorción de la radiación. Suele emplearse para determinar la composición química de las sustancias identificando grupos funcionales e iones metálicos. En el caso del quitosano, se emplea para evaluar el grado de desacetilación (Czechowskabiskup et al. 2012), o para determinar el contenido de moléculas orgánicas (Esmaeili & Asgari 2015) o nanopartículas metálicas (Moharram et al. 2014) en un composite de base quitosano. FT-IR. La espectroscopía infrarroja (IR), consiste en la cuantificación de las oscilaciones de los átomos de una molécula tras la absorción de energía emitida en un rango de frecuencia de 12 800 a 4 000 cm-1 en el IR cercano, de 4 000 a 400 cm-1, en el IR medio (más utilizado) o 400 a 50 cm-1 en el IR lejano. La energía no absorbida por la muestra se capta en un colector y la transformada de Fourier posibilita la formación un espectro interpretable. El espectro vibracional de una molécula se considera una propiedad física única, por tanto es una “huella dactilar” que la identifica. A través de FTIR se ha podido comprobar la interacción entre el quitosano y los polifenoles del propóleo, que se unen a través de enlaces de hidrógeno (Siripatrawan & Vitchayakitti 2016). ATR – FTIR. La reflectancia total atenuada (ATR por sus siglas en inglés) es una técnica acoplada a un espectrofotómetro FTIR, para un análisis rápido de las muestras en estado sólido, gel o líquido. Consiste en la emisión de un haz de IR que se desplaza por un cristal ópticamente denso y con alto índice de refracción, en un ángulo determinado; la reflectancia interna en el cristal crea una onda evanescente que se extiende hasta hacer contacto con la muestra ubicada en la superficie. En las regiones del espectro IR donde la muestra absorbe energía, la onda evanescente se atenúa, el haz atenuado vuelve al cristal, sale a continuación por el extremo opuesto a la emisión y se dirige al detector para generar un espectro de IR. Esta técnica ha sido utilizada para evaluar el rol de los grupos funcionales del quitosano (OH y NH) en la formación de enlaces de hidrógeno, para la preparación de un film con nanoarcillas (Branca et al. 2016). THz – TDS. La radiación de terahercios medida tiempo-dominio (THz–TDS por sus siglas en inglés) es una técnica de espectroscopia en la cual las propiedades de la muestra son estudiadas por la irradiación de ondas desde 100 µm a 1 mm de longitud. Esta provee más información que la espectroscopía por transformada de Fourier, que es solo sensible a la amplitud. Debido a que se conoce el tiempo-dominio, la frecuencia-dominio de la señal de THz está disponible, así el efecto de distorsión de la difracción se puede mitigar y la resolución de las imágenes de THz puede ser mejorada (Ahi 2018). | Introducción General Biocomposites antifúngicos de base quitosano para la protección de especies agrícolas, forestales y de madera | 17 DFT y GPU. La química cuántica computacional es una herramienta muy valiosa para la interpretación de espectros Far FT-IR debido a la complejidad de los modos de vibración asociados. Los métodos Ab initio y la teoría funcional de la densidad (DFT) son muy precisos en la predicción de vibraciones moleculares (Scott & Radom 1996). Sin embargo, los cálculos de estado sólido sobre grandes sistemas supramoleculares generalmente son necesarios debido a la relevancia de las interacciones de largo alcance, lo que limita el uso de estos métodos computacionalmente costosos. Por otro lado, los métodos semiempíricos (Coolidge et al. 1991), especialmente con las implementaciones paralelas desarrolladas recientemente para multiprocesadores de memoria compartida y arquitecturas de unidades de procesamiento de gráficos GPU (Maia et al. 2012), permiten el estudio de las vibraciones en grandes cristales moleculares en tiempos de cálculo reducidos. XRD. La difracción de rayos X (XRD por sus siglas en inglés) se utiliza para determinar la disposición de los átomos en un cristal, consiste en la emisión e incidencia de un rayo X sobre el cristal, el cual se difracta en muchas direcciones específicas. Desde estos haces difractados, se puede producir una imagen tridimensional de la densidad de los electrones dentro del cristal. Después, a partir de la densidad electrónica, se pueden determinar las posiciones medias de los átomos, así como otra información estructural. En el caso de polímeros como el quitosano, por sus características anisotrópicas un patrón de cristalinidad es difícilmente obtenido, por tanto se deduce que es un material amorfo (Kim 2014). El método de Debye-Scherrer en XRD es utilizado para estudiar la estructura de sustancias finamente cristalinas, por ejemplo, la formación de nanopartículas de plata utilizando quitosano en el proceso de síntesis (Wang et al. 2015). Técnicas microscópicas SEM. La microscopía electrónica de barrido (SEM por sus siglas en inglés) es una tecnología aplicada a la caracterización morfológica de las muestras capaz de producir imágenes de alta resolución. Consiste en la interacción electrón-materia, un haz de electrones acelerados en un campo eléctrico es emitido y enfocado a través de lentes hacia la muestra, cuando el haz incide sobre ella se producen interacciones entre los electrones del haz y los átomos de la muestra; por ejemplo, electrones secundarios desprendidos de la muestra pueden salir disparados tras la colisión, siendo estos los electrones detectados generalmente para la formación de las imágenes. Las micrografías de SEM se utilizan frecuentemente en los estudios de síntesis de materiales compuestos a partir de quitosano para determinar la micromorfología de los mismos (Torabi et al. 2016; Fortunati et al. 2017). TEM. Microscopio electrónico de transmisión (TEM por sus siglas en inglés). Este tipo de microscopio aunque aplica el mismo principio que un SEM (un haz de electrones acelerados disparados hacia la muestra) tiene mejores prestaciones, pues tiene la capacidad de aumentar hasta 1 millón de veces el tamaño de la muestra, debido a que la imagen se | Introducción General Biocomposites antifúngicos de base quitosano para la protección de especies agrícolas, forestales y de madera | 18 genera directamente de los electrones que la atraviesan, por lo cual esta debe tener de un espesor muy fino. La morfología de la superficie y la forma de varios nanomateriales pueden obtenerse de esta forma (Gu et al. 2014; Saharan et al. 2015). Tabla 2. Técnicas principales utilizadas para la caracterización de materiales compuestos basados en quitosano. Compuesto FTIR UV-Vis TEM SEM XRD Referencia Clorhidrato de quitosano x x Fortunati et al. 2017 Quitosano-piridina x x x Jia et al. 2016 Quitosano-sílica x x x Dabóczi et al. 2016 Quitosano-acrílico x x Torabi et al. 2016 Quitosano - nanopartículas de vidrio bioactivo x x x x Pourhaghgouy et al. 2016 Quitosano-óxido de grafeno-trientina x x x Ge and Ma 2015 Quitosano-alginatoaceites esenciales x x Natrajan et al. 2015 Quitosano-Vainillina x x x Stroescu et al. 2015 Quitosano-Cu NPs x x x Saharan et al. 2015 Quitosano-aceite de neem-óxido de zinc x x x Sanuja et al. 2015 Quitosano-Ag NPs x x x Moharram et al. 2014 Quitosanopolicaprolactona-Ag NPs x x x x Gu et al. 2014 Quitosano-isicianato x Gallego et al. 2013 Quitosano-cloroquinolina x x x Kumar et al. 2011 1.4 Composites de quitosano para la sanidad agroforestal El quitosano y sus derivados han sido estudiados con el objetivo de ser aplicados potencialmente en el área agrícola, a través de dos usos generales: como antimicrobiano y como estimulante vegetal; ambas propiedades, son conferidas por el carácter catiónico de sus moléculas y la reactividad de sus grupos funcionales. Como se mencionó anteriormente, la capacidad antimicrobiana del quitosano se ha utilizado para el control de enfermedades causadas por hongos fitopatógenos. Los formas de ser aplicado varían según el objetivo de la aplicación, de tal forma que puede encontrarse como recubrimiento de semillas, tratamiento foliar, recubrimiento de frutos post cosecha, enmienda de suelos, etc. | Introducción General Biocomposites antifúngicos de base quitosano para la protección de especies agrícolas, forestales y de madera | 19 1.4.1 Aplicaciones de quitosano Estimulante vegetal El quitosano puede ser usado en las plantas como estimulante, elicitor o inductor de crecimiento y resistencia a enfermedades causadas por agentes bióticos o abióticos. La aplicación del quitosano favorece la germinación de las semillas, el vigor de las plantas y el rendimiento productivo (Ramirez et al. 2010). Se ha comprobado que al interior de las plantas, el quitosano y sus derivados pueden activar enzimas encargadas de la degradación y movilización de nutrientes, incluso pueden activar hormonas de crecimiento como la auxina y la citoquinina, entre otros mecanismos involucrados en el crecimiento (Kumaraswamy et al. 2018). Además puede regular el sistema inmune de las plantas, incrementando la producción de enzimas antioxidantes tales como la superóxido dismutasa, catalasa y peroxidasa, las cuales atacan a las especies reactivas de oxígeno, mejorando la tolerancia a los factores de estrés (Zeng and Luo 2012). La aplicación de quitosano en plantas de interés agrícola como arroz, soja, tomate y lechugas, se ha evaluado en las primeras etapas de crecimiento y se han encontrado incrementos en el tamaño y peso de las hojas, al aplicarse en solución a la zona foliar (Chibu and Shibayama 2003). En pruebas de campo se ha comprobado que plántulas de trigo, germinadas de semillas recubiertas con quitosano, han presentado mejores características físicas (tasa de germinación, contenido de biomasa, actividad radicular, rendimiento), así como fisiológicas (contenidos estables de enzimas y sales) bajo condiciones de sequía (Zeng and Luo 2012). También se han realizado aplicaciones de quitosano en especies forestales como pino silvestre en vivero, la dosificación en spray de quitosano soluble en agua, a pesar de que no incrementó la germinación, sí mejoró la longitud de las plántulas y el contenido de biomasa en la parte aérea y raíz (Aleksandrowicz-trzcińska et al. 2015). Como reportan Khan et al. 2003, también se ha comprobado el aumento de la actividad enzimática en diferentes cultivos agrícolas como la soja, con la aplicación de oligómeros de quitosano de distintos tamaños, donde la fenilalanina amonio liasa y la tirosina amonio liasa incrementaron su actividad, estas dos son enzimas precursoras de metabolitos secundarios como la lignina, flavonoides y fitoalexinas, que desempeñan un papel clave en la interacción planta-patógeno. Control antifúngico El quitosano posee por sí mismo propiedades para combatir un amplio espectro de organismos fitopatógenos (Katiyar et al. 2014). El contacto directo actúa en contra de la superficie celular de los hongos ejerciendo alguno de los mecanismo de acción descritos en el apartado 1.1.3, la lisis de la membrana plasmática o la interrupción de síntesis de proteína al interior de las células. | Introducción General Biocomposites antifúngicos de base quitosano para la protección de especies agrícolas, forestales y de madera | 20 En evaluaciones in vitro adicionando diferentes tipos de quitosano en el medio de cultivo se ha demostrado un efecto fungicida o inhibidor del crecimiento de micelio de hongos que causan pudrición de vegetales, como Aspergillus niger, Alternaria alternata, Rhizopus oryzae (Ziani et al. 2009); pudrición de semillas, como Aspergillus flavus, Rhizoctonia solani (Kaur et al. 2012); o pudrición de madera, como Leptographium procerum y Sphaeropsis sapinea (Torr et al. 2005), entre muchos otros, para los cuales se han reportado las concentraciones mínimas de inhibición de crecimiento (MIC). Por otra parte los estudios in vivo, demuestran cómo la protección de semillas o frutos con recubrimientos semipermeables de materiales de base quitosano, son capaces de aislar al producto del ataque de los hongos extendiendo su tiempo de almacenaje. Por ejemplo, en semillas de Jatropha curcas se ha probado un recubrimiento de quitosano de bajo PM, encontrando que más de un 60% de semillas germinadas fueron protegidas del ataque del hongo (Pabón-Baquero et al. 2015). Un recubrimiento de aloe vera con quitosano extendió la vida post cosecha de mora azul más de 5 días, el recubrimiento consiguió aislar a la fruta del ataque de Botrytis cinerea (Vieira et al. 2016). También se han encontrado resultados efectivos en Carica papaya al aplicar un film de quitosano y almidón, tras 15 días de almacenamiento a temperatura ambiente, la calidad en la piel y pulpa de la papaya se mantuvieron intactas, mientras que sin el film solo se mantienen 5 días (Escamilla-García et al. 2018). Más pruebas en mango (Chowdappa et al. 2014), kiwi y lechuga (Fortunati et al. 2017), zanahorias, manzanas, fresas, frambuesas, tomates (Gutiérrez-Martínez et al. 2016) y otros cultivos agrícolas tratados con recubrimientos basados en quitosano han registrado efectos exitosos preservándolas de los efectos negativos causados por hogos fitopatógenos. 1.4.2 Enfermedades agrícolas y forestales Las enfermedades causadas por hongos fitopatógenos son las principales causantes de grandes pérdidas económicas asociadas a la producción agrícola y forestal. En la tabla 3 se describen algunos de los hongos más devastadores de diferentes cultivos de interés económico y ecológico a nivel mundial como el café, la madera de chopo y diversidad de coníferas. Tabla 3. Algunos de los hongos fitopatógenos de especies agrícolas y forestales más importantes a nivel mundial por los daños que causan Especie/División Localización Hospedero Patología Referencia Fusarium circinatum Ascomiceto Nivel mundial Coníferas. Más de 60 especies de Pinus susceptibles Chancro de coníferas. La más grande amenaza de bosques de pinos a nivel mundial MartínezÁlvarez 2015 Diplodia pinea Ascomiceto Nivel mundial Coníferas. Chancro de coníferas. Causa chancros en el tronco de los árboles, desecación MartínezÁlvarez 2015 | Introducción General Biocomposites antifúngicos de base quitosano para la protección de especies agrícolas, forestales y de madera | 21 Especie/División Localización Hospedero Patología Referencia Pinus radiata la especie más susceptible en los brotes, inhibición de la germinación Gremmeniella abietina Ascomiceto Europa, Norte América y Japón Coníferas. Como picea, abeto, alerce, pino y enebro Acículas secas, defoliación de la copa, distorsión de ramillos terminales y eventualmente la muerte de algunos pies Romeralo 2015 Cryphonectria parasítica Ascomiceto Asia, Europa y Norte América Especies del género Castanea Chancro del castaño. Chancros en el tronco, ramas o brotes que obstruyen la circulación de savia, desecando los brotes o ramas encima de la lesión Zamora et al. 2012 Heterobasidium annosum Basidiomiceto Hemisferio norte Coníferas Pudrición de las raíces y el pie de coníferas. Presenta crecimiento anormal de las acículas, corteza amarilla y pálida o marchitez general Prieto-Recio et al. 2014 Hemileia vastatrix Basidiomiceto Nivel mundial Especies del género Coffea. Principalemente cultivos de C. arabica Roya de las hojas del café. Manchas color naranja intenso sobre las hojas, que causan su pudrición. Cristancho et al. 2012 Trametes versicolor Basidiomiceto Nivel mundial Madera estructural. Diferentes especies (duras y blandas) Pudrición blanca de la madera, la degradación más importante y extendida Spavento 2015 Phytophthora ×alni Oomiceto Europa Alisos Muerte del aliso común. Desecación del árbol, hojas amarillentas en la copa, exudados enmohecidos a la altura del cuello y parte baja del tronco Érsek and Nagy 2008 Phytophthora camvibora Oomiceto Europa Castaño, hayas y otras maderas duras Tinta del castaño. Coloniza las raíces más pequeñas e infecta el tronco consiguiendo matar al árbol rápidamente Jung et al. 2005 Phytophthora plurivora Oomiceto Austria, Alemania, Rumania y España Alisos Chancro y pudrición del tronco. Afecta la parte aérea del árbol, cuello y parte baja del tronco, aunque se encuentra en el suelo, en la zona radicular Haque et al. 2014 | Materiales y Métodos Biocomposites antifúngicos de base quitosano para la protección de especies agrícolas, forestales y de madera | 28 Equipos: - Espectrómetro THz-TDS Tera K15 de Menlo para el análisis de NAG (Art. 1) - Espectrómetro FTIR Nicolet iS50 de Thermo Scientific equipado con un sistema de Reflexión Total Atenuada (ATR) de diamante, para análisis de estructura química de la NAG, composites y degradación de la madera (Art. 1, 2, 3 y 6) - Espectrómetro UV-Vis Shimazdu UV-2450 para el análisis de la nanoplata (Art. 2) - Difractómetro de Rayos X Bruker D8 Advance Bragg-Brentano para caracterización de composite (Art. 2) - Microscopio Electrónico de Transmisión (TEM) JEOL JEM-FS2200 HRP equipado con una sonda INCA Energy TEM 250 EDS de Oxford Instruments para caracterización de composite (Art. 2) - Microondas Milestone Ethos-One para síntesis de composites (Art. 2) - Sonicador model CSA 20-S500 para síntesis de composites (Art. 2, 3, 5 y 6) - Espectrómetro UV-Vis Multiscan Go Microplate de Thermo Scientific para el análisis de TPC y RSA en plántulas (Art. 5) - Microscopio óptico de transmisión Leica DMLM para análisis de degradación de la madera (Art. 6) Softwares: - PM6 software para análisis molecular de NAG (Art. 1) - R software, para análisis estadísticos (Art. 3, 4, 5 y 6) 4.2 Síntesis de composites Materiales individuales: - Quitosano de medio peso molecular (QMPM) disuelto en ácido acético (1%) (Art. 2, 3, 4, 5 y 6) - Oligómeros de quitosano (OQ) obtenidos por degradación oxidativa con H2O2 (Art. 2, 3, 4, 5 y 6) - Extracto etanólico de propóleo (EEP) en solución hidroalcohólica 7:3 v/v (Art. 3, 4, 5 y 6) - Nanopartículas de plata (nAg) por reducción con citrato de sodio y NaBH4 con sonicación (Art. 2) - Nanopartículas de plata con quitosano como agente reductor y estabilizante en autoclave ( Art. 3) - Nanopartículas de plata por reducción con citrato de sodio en tratamiento térmico (Art. 6). | Materiales y Métodos Biocomposites antifúngicos de base quitosano para la protección de especies agrícolas, forestales y de madera | 29 Composites: - Composite de OQ-rGO-nAg por microondas a 60 y 120 °C (Art. 2) - Mezclas binarias y ternaria de OQ, P y nAg por cortos ciclos de sonicación, mezclando las soluciones individuales a las concentraciones deseadas (Art. 3, 4, 5 y 6) 4.3 Pruebas in vitro - Concentración mínima inhibitoria (MIC) de QMPM, OQ, EEP, nAg, y mezclas obtenidas por pruebas de difusión en agar para T. versicolor (Art. 6) - Efectividad de OQ, EPP, nAg y sus combinaciones contra los ocho patógenos forestales, evaluada por difusión en agar a las MIC de la prueba anterior (Art. 3) - Efecto del OQ, EPP y OQ-EPP en la germinación de las urenidiosporas de H. vastatrix evaluado en medio líquido (Art. 4). 4.4 Pruebas in vivo - Efecto del OQ, EEP y OQ-EPP, en el desarrollo de H. vastatrix evaluado sobre discos de hojas de café en tres distintas aplicaciones (Art. 4) - Efecto de QMPM, OQ, EEP, QMPM-EPP y OQ-EPP evaluado como recubrimientos bioactivos de semillas de P. silvestrys y P. radiata (Art. 5) - Efecto del QMPM, OQ, OQ-EEP y OQ-EEP-nAg evaluado como protector superficial de madera de Populus spp (Art. 6) 4.5 Análisis estadístico - Análisis de varianza (ANOVAs) y de comparación múltiple (Art. 3, 4, 5 y 6) - Procedimientos generalizados de Welch (Art. 3, 6) - Pruebas de Kruskal-Wallis (Art. 4) - Prueba Chi cuadrado (χ²), Kaplan-Meier y de Tukey (Art. 5) - Prueba de Shapiro-Wilks, de Bartlett y Bootstrapping (Art. 6) Biocomposites antifúngicos de base quitosano para la protección de especies agrícolas, forestales y de madera | 30 5. RESULTADOS PRINCIPALES En este apartado se presentan los resultados más destacados de la revisión y de cada uno de los artículos que componen el compendio de publicaciones, organizados por el tipo de información que aportan: estudios teóricos y estudios prácticos. Por ejemplo, el estudio de la estructura y formación de nuevos nanocompuestos a partir de quitosano, el análisis molecular del monómero constituyente de sus cadenas poliméricas, la NAG; la síntesis y caracterización de composites basados en oligómeros de quitosano, ya sea con rGO, EEP o nAg; así como los datos descubiertos en las aplicaciones in vitro: las MIC para T. versicolor, la efectividad de las combinaciones de los materiales antifúngicos individuales para ocho patógenos forestales y la efectividad de OQ y EEP -solos y en mezclaen la germinación de esporas de H. vastatrix. Finalmente, se presenta una de las aportaciones más importantes de esta Tesis en el ámbito de sus aplicaciones directas en la ingeniería agrícola y forestal: el efecto de los productos contra enfermedades como la roya de las hojas del café, el chancro de los pinos o la pudrición blanca de la madera evaluados in vivo. 5.1 Análisis molecular, síntesis y caracterización de composites Nanomateriales basados en quitosano (Review) El carácter alcalino del quitosano, debido a la presencia de grupos amino, especialmente en los oligómeros, brinda una mayor tendencia para formar enlaces con biomoléculas como la nisina o liposomas; con extractos naturales como propóleo, tomillo, canela o vainillina; incluso con minerales, metales o productos de síntesis como sílice, nanoplata o polivinilpirrolidona (PVA). Estos nuevos materiales pueden sintetizarse a través métodos bilógicos como hidrólisis enzimática; físicos como microondas, ultrasonidos o radiación; y químicos como copolimerización, hidrólisis ácida, catálisis, etc. Todos estos materiales pueden ser de gran utilidad en aplicaciones para biomedicina, alimentos, medio ambiente, preservación del patrimonio y agricultura, principalmente. N-acetil-D-glucosamina (Art. 1) El espectro infrarrojo lejano de la N-acetil-D-glucosamina (NAG) generado por Far-FTIR y THz-TDS muestra un pico dominante a 60 cm-1 y dos más, a 45 y 72 cm-1 aunque de mucha menor intensidad en la zona de los THz. En la zona de FTIR se encontraron resonancias más definidas e intensas, las cuales se corresponden cercanamente a las frecuencias teóricas encontradas a partir del método PM6, un método semiempírico de química cuántica. El espectro vibracional obtenido muestra bandas adicionales no registradas en la literatura. | Resultados Principales Biocomposites antifúngicos de base quitosano para la protección de especies agrícolas, forestales y de madera | 31 Síntesis y caracterización de rGO-OQ-nAg (Art. 2) El material compuesto de óxido de grafeno reducido (rGO), oligómeros de quitosano (OQ) y nanopartículas de plata (nAg), obtenido exitosamente por un sencillo proceso de síntesis asistido por microondas a 60°C y 120 °C, fue caracterizado por FTIR, UV-vis, XRD y TEM. A través de cambios en las bandas de absorción del espectro FTIR de los OQ se observa la presencia del rGO y de la nAg en el material compuesto. Por ejemplo, un pico intenso a 1636 cm-1 y otro más débil 1412cm-1, denotan enlaces C=O y C-O de grupos carboxilo o alcoxi y la amida. La espectroscopía UV-vis revela más claramente la formación del material compuesto, debido a que el plasmón de la nanoplata, registrado a 420 nm, permanece en los composites, aunque en menor intensidad. El estudio por XRD muestra un espectro donde se identifican los tres componentes del material, un banda a 2θ ≈ 21° pude asignarse a los OQ, a 24° al rGO y otra bastante intensa a 38° corresponde a la nAg de acuerdo a la comparación con un patrón de referencia. En las micrografías por TEM se pudo observar que las Ag NPs son esféricas, tienen un diámetro entre 10 y 30 nm y se observan incrustadas en la matriz de hojas de rGO y OQ. La temperatura no muestra diferencias significativas en las características de los composites, por tanto 60°C son suficiente para el proceso de síntesis. Caracterización de OQ, OQ-EEP, OQ-EEP-nAg (Art. 3) La interacción de los oligómero de quitosano con los grupos funcionales del propóleo y la quelación de las nanopartículas de plata, fue evaluada a través de FTIR, comparando el espectro individual de OQ, el de la mezcla binaria OQ-EEP y la ternaria OQ-EPP-nAg. El primer espectro (OQ) mostró las bandas características de absorción del quitosano (a 3256 cm-1 las bandas sobrelapadas del O-H y N-H, entre otras variadas vibraciones entre C y O en la zona de la huella dactilar de 1600 cm-1 a 800 cm-1). Los enlacen entre OQ y EPP se evidenciaron con picos más altos en la huella dactilar y un desplazamiento de 1257 cm-1 a 1263 cm-1 asociado al ν(CΦ-O) debido a enlaces por puentes de hidrógeno. Respecto a la nAg se encontraron ligeros decrecimientos en algunas bandas, sin embargo, no se observó un esperado desplazamiento en un pico asociado a la amina del OQ, lo que hubiera indicado un fuerte enlace, por el contrario se evidencia una débil interacción. 5.2 MIC y efectividad in vitro Concentración mínima inhibitoria (Art. 6) El trabajo experimental de esta tesis respecto el efecto antifúngico inició con la evaluación de la concentración mínima inhibitoria (MIC), es decir, la concentración que alcanza el 100% de inhibición del crecimiento del micelio, de quitosano de mediano PM (QMPM), oligómeros de quitosano (OQ), extracto etanólico de propóleo (EEP) y nanopartículas de plata (nAg), individualmente y en mezclas binarias y ternarias contra el hongo T. versicolor. | Resultados Principales Biocomposites antifúngicos de base quitosano para la protección de especies agrícolas, forestales y de madera | 32 La MIC para OQ fue de 3 mg·mL-1, para EPP de 0,5 mg·mL-1 y para nAg de 3 µg·mL-1. En el caso de QMPM la concentración más alta aplicada (10 mg·mL-1) inhibió el 95,5 % del crecimiento del micelio. Las mezclas binarias de OQ, EPP y nAg mostraron un efecto mejorado respecto a los productos individuales, pues se alcanzó el 100% de inhibición con concentraciones de 2,0 y 0,2 mg·mL-1 para OQ-EEP (respectivamente) y 2 µg·mL−1 para nAg en la mezcla ternaria. Efecto antifúngico y antioomicetos contra patógenos forestales (Art. 3) Se evaluaron in vitro soluciones de OQ (1 mg·mL-1), EEP (0,1 mg·mL-1) y nAg (1 µg·mL-1), individualmente y en mezclas, para controlar el crecimiento de diferentes hongos y oomicetos fitopatógenos de árboles de interés forestal. Estas dosis fueron elegidas a partir del análisis de la MIC del estudio anterior. La solución ternaria no presentó un efecto mejorado sobre las mezclas binarias OQ-EEP y OQ-nAg, las cuales tuvieron el mayor efecto antifúngico contra los hongos Fusarium circinatum (con 82% de inhibición) y Diplodia pinea (con 77% de inhibición), respectivamente. La solución de OQ por sí misma también resultó muy efectiva contra los hongos Gremmeniella abietina, Cryphonectria parasitica y Heterobasidion annosum, causando una inhibición del 78%, 86% y 93% respectivamente, además de un 100% en el oomiceto Phytophthora cambivora. Por otro lado, el EEP inhibió totalmente (100%) el crecimiento de Phytophthora ×alni y Phytophthora plurivora. Por su parte, la aplicación de nAg individualmente redujo el crecimiento micelial de F. circinatum y D. pinea, sin embargo, las mezclas binarias y ternaria con nAg, tuvieron un efecto contraproducente en la actividad antifúngica y anti-oomiceto sobre algunos patógenos. Efecto sobre la germinación de esporas de H. vastratrix (Art. 4) Soluciones acuosas de OQ y EEP, individualmente o como mezclas binarias, se probaron contra Hemileia vastatrix, el hongo que causa la roya de las hojas de café, la peor enfermedad del café a nivel mundial. En este estudio fueron aplicados únicamente los OQ y EEP, debido a los mejores resultados observados en el análisis anterior. Los resultados muestran una excelente actividad antifúngica de los OQ a 2 mg·mL-1, pues la germinación de esporas de H. vastatrix fue inhibida en un 99,5%. En el caso del EEP, con una dosis 10 veces menor (0,2 mg·mL-1) el resultado fue la mitad de efectivo que los OQ, con 54,4% de inhibición. De tal forma que en la mezcla de OQ-EPP se observa una ligera reducción del efecto de los oligómeros solos, con 96,6% de inhibición. 5.3 Control de enfermedades de especies agrícolas, forestales y madera Aplicación en hojas de café (Art. 4) La efectividad de las soluciones de OQ, EEP y su mezcla para evitar los daños que causa la enfermedad de la roya del café (CLR) fue evaluada sobre discos de hojas con ensayos en | Resultados Principales Biocomposites antifúngicos de base quitosano para la protección de especies agrícolas, forestales y de madera | 33 tres diferentes tratamientos. En el primer tratamiento (inoculación del patógeno y 24 horas después aplicación de soluciones) el patógeno causó daños sobre un 12% del disco y no se observaron diferencias significativas con los tratamientos. En el segundo tratamiento (aplicación de soluciones y 24 horas después inoculación) se encontraron excelentes resultados, las soluciones de OQ, EEP y OQ-EEP presentan una protección total de los discos, pues los daños causados por el hongo 30 días después de la inoculación representan solo 0,2%. En el tercer tratamiento (aplicación de soluciones a la vez que la inoculación) también se encontraron resultados positivos con la aplicación de las soluciones respecto al control, el cual registra la afectación más alta (20% del disco). En este caso, el EEP fue menos efectivo que los OQ y la mezcla. Recubrimientos bioactivos (Art. 5) En este estudio, semillas de P. sylvestris y P. radiata fueron recubiertas con tratamientos de QMPM, OQ, EEP y las mezclas de ambos quitosanos con EPP, para evaluar su potencial como protectores de estas semillas ante la presencia de F. circinatum en el sustrato de cultivo. En una evaluación previa, en sustrato no inoculado, se encontró que los recubrimientos no afectan la germinación de las semillas, excepto por los recubrimientos de QMPM en P. sylvestris y de OQ-EEP en P. radiata, que la reducen ligeramente. Todos los recubrimientos presentaron un impacto positivo en P. sylvestris, especialmente QMPM, OQ y QMPM-EEP con más de 50% de sobrevivencia de plántulas germinadas de semillas recubiertas respecto a las no protegidas, las cuales murieron a los 40 días después de la inoculación. Este efecto se comprobó en el contenido total de polifenoles (TPC) y en la actividad antioxidante (RSA) de las plántulas, las cuales presentaron tasas similares a plántulas sanas (de semillas no inoculadas). En el caso de P. radiata la eficacia de los tratamientos fue limitada, aunque se sabe que es la especie más susceptible a este patógeno. Protección de madera (Art. 6) Los compuestos de QMPM, OQ, OQ-EEP y OQ-EEP-nAg se probaron en pequeños bloques de madera de chopo como protectores superficiales durante 30 días de exposición a T. versicolor, el hongo causante de la pudrición blanca. Con una toma de muestra cada 5 días, se fue registrando la pérdida de peso de los bloques, que alcanzaron un 42,3% en la madera no protegida. Por el contrario, todos los tratamientos exhibieron una protección debido a que la pérdida de peso fue menor, 11,4% en QMPM, 26,9% en OQ, 32,8% en OQ-P y 39,9% en OQ-EEP-nAg, de tal forma que el composite ternario es el único que no presenta diferencias significativas. El seguimiento de la degradación de la madera por microscopía óptica y espectroscopía FTIR, también evidenció que el QMPM es el mejor agente protector, probablemente debido a su mayor viscosidad y propiedades de adhesión. Biocomposites antifúngicos de base quitosano para la protección de especies agrícolas, forestales y de madera | 34 6. DISCUSIÓN GENERAL El quitosano se considera uno de los polímeros de origen natural más prometedores en la preparación de nuevos materiales nanocompuestos. Como se ha reportado en la revisión bibliográfica, el quitosano y sus oligómeros pueden enlazarse a diversas especies de origen natural o de síntesis; como propóleo (Matei et al. 2015), vainillina (Stroescu et al. 2015), aceites esenciales de canela (Ojagh et al. 2010) etc., o nanoplata (Venkatesham et al. 2012), cobre (Xue and Wilson 2016), sílice (Dabóczi et al. 2016) o PVA (Tahtat et al. 2011), entre otros. Además, la versatilidad de los materiales derivados del quitosano les permite ser procesados en geles (Jun et al. 2010), membranas (Santos et al. 2013), nanofibras (Pillai et al. 2009), esferas (Benamer et al. 2011), micropartículas, nanopartículas y bloques (Shukla et al. 2013) a través de varios métodos de síntesis que involucran procesos biológicos, químicos y físicos, por ejemplo, hidrólisis enzimática (Zou et al. 2016), co-polimerización (Thakur and Thakur 2014), irradiación gama (Taşkin et al. 2014), microondas (Ge and Ma 2015) o técnicas de ultrasonidos (Ho et al. 2016). Lo anterior, denota que actualmente existe un interés sustancial en la investigación para el desarrollo de nuevos nanomateriales de origen renovable, como los basados en quitosano. El análisis molecular de la NAG reportado en el Artículo 1, ha sido realizado por primera vez por un método experimental a partir del espectro infrarrojo lejano (obtenido por Far-FTIR y THz-TDS), así como por un método teórico semiempírico de química cuántica (PM6), el cual mostró bandas adicionales en el espectro vibracional, no registradas en un estudio similar realizado por Kovacs et al. (2008). En comparación con los estudios previos basados en ab inition y la teoría funcional de la densidad (DFT), el enfoque semiempírico elegido en este trabajo, adecuado para la aceleración de la unidad de procesamiento gráfico (GPU) y de múltiples núcleos en paralelo, permite un estudio completo utilizando condiciones de frontera periódicas y no solo aproximaciones dentro de un tiempo de cálculo limitado. El conocimiento de las características vibracionales de esta molécula puede ayudar a predecir modos de acción antifúngicos específicos en estudios de estructura-actividad del quitosano. La síntesis del material compuesto por rGO-OQ-nAg reportada en el Artículo 2, asistida por microondas y su caracterización, mostraron un método sencillo y adecuado para el autoensamblaje del rGO en la matriz polimérica de los OQ y la estabilización de la nAg, pues nanoagrados de este metal se observaron anclados uniformemente en las hojas de rGO funcionalizadas con OQ. Además, la principal ventaja encontrada en este composite es que presenta una forma sólida cristalina de textura grafénica y no de hidrogel, lo cual facilitaría y ampliaría el rango de sus aplicaciones (Jiao et al. 2015; Marta et al. 2015). La caracterización por FTIR reportada en el artículo 3 de los composites OQ, OQ-EEP y OQEEP-nAg reveló la interacción entre los grupos funcionales del OQ y el EEP como se observa en estudios similares (Siripatrawan & Vitchayakitti 2016), no obstante, mostró una débil | Discusión General Biocomposites antifúngicos de base quitosano para la protección de especies agrícolas, forestales y de madera | 35 interacción con la nAg, a diferencia de otros trabajos (Wang et al. 2015), probablemente debido a las bajas dosis de nAg utilizadas en este estudio. La MIC reportada en el artículo 6 para T. versicolor muestra una mayor actividad antifúngica in vitro de OQ vs QMPM, así como un mejor desempeño de las mezclas binarias en comparación con los productos individuales (la inhibición completa del crecimiento del micelio se logró a concentraciones de 2 mg·mL-1 para OQ, 0.2 mg·mL-1 para EEP y 2 µg·mL-1 para nAg para cualquiera de las mezclas binarias). No obstante, no se encontraron mejoras significativas asociadas con el compuesto ternario sobre sus contrapartes binarias para este hongo; probablemente debido a que las nanopartículas metálicas tienden a asociarse fuertemente a moléculas orgánicas impidiendo su difusión al medio. Las MIC determinadas en este estudio se encuentran dentro de un rango de concentraciones semejantes a las dosis aplicadas en otros trabajos sobre la actividad antifúngica del quitosano (Badawy & Rabea 2009; Younes et al. 2014), el propóleo (Pastor et al. 2010; Ali et al. 2013) y la nanoplata (Wang et al. 2015; Kim et al. 2018), como de sus mezclas. Lo que denota la necesidad de evaluar la dosis óptima de cada producto según la especie. La actividad contra microorganismos patógenos forestales reportada en el artículo 3, muestra que todas las soluciones individuales y las mezclas demostraron capacidad de inhibición del crecimiento micelial contra alguno de los microorganismos estudiados. Sin embargo, esta actividad antifúngica y anti-oomicetos mostró una dependencia del tipo particular de microorganismo. El tratamiento con la mayor actividad registrada fueron los OQ, que por sí solos presentaron tasas de inhibición del 78% al 100% contra 4 de los microorganismos, este efecto ha sido estudiado en otros hongos (Ahmed et al. 2001; Avelelas et al. 2014; Qiu et al. 2014; Cobos et al. 2015), no obstante, las dosis aplicadas pueden ser superiores a 1 mg·mL-1 utilizada en este estudio. La aplicación del EEP fue exitosa contra dos oomicetos (P. ×alni y P. plurivora), inhibió el 100% del crecimiento con solo 0,1 mg·mL-1; la actividad antibacteriana del propóleo es bien conocida en medicina tradicional, sin embargo, como antifúngico se ha estudiado contra pocas especies (Iturritxa et al. 2013). Por otra parte la nanoplata sí ha sido estudiada como antifúngica contra diversos hongos (Narayanan and Park 2014; Mahdizadeh et al. 2015), aunque en el presente estudio sólo presentó efectos significativos contra F. circinatum y D. pinea. La capacidad antifúngica y anti-oomicetos de los OQ fue incrementada por las mezclas binarias en algunos casos, como OQ-EPP en F. circinatum y OQ-nAg en D. pinea, que alcanzaron tasas de inhibición alrededor de 80%. Sin embargo, la adición de un tercer componente no presentó un efecto mejorado sobre estas; contrario a la actividad contra Diplodia seriata (Matei et al. 2015) o Bipolaris Oryzae (Araujo-Rufino et al. 2016) donde el mejor efecto antifúngico fue observado al combinar OQ, EPP y nAg, aunque a dosis superiores a las aplicadas en este trabajo. La evaluación in vitro del artículo 4, donde la capacidad de los OQ para inhibir la germinación de esporas de hongos fitopatógenos, se evidencia una vez más en este estudio | Discusión General Biocomposites antifúngicos de base quitosano para la protección de especies agrícolas, forestales y de madera | 36 contra H. vastatrix, donde se ha encontrado una excelente opción para el tratamiento de la enfermedad causada por este hongo en las hojas de café. En un estudio llevado a cabo por Rahman et al. (2015), se ha observado una hinchazón de las esporas además de inclusiones granulares en el citoplasma de las células tras el contacto con oligómeros de quitosano, lo cual genera una interrupción del desarrollo del tubo germinativo, es decir, la inhibición de la germinación. El propóleo, otro producto de origen natural, también mostró una alta actividad antifúngica considerando la baja concentración utilizada, como en el caso de otros estudios (Silva-Carvalho et al. 2015) su efecto podría mejorarse aumentando la dosis de aplicación. La combinación de ambos productos (OQ-EPP) no presentó mejores resultados que el uso de OQ individualmente. En el artículo 4 también se evalúa, la aplicación de las soluciones de OQ, EEP y su mezcla después de la inoculación del patógeno sobre los discos de hojas del café donde no causó un efecto positivo, debido a que la infección inicial del hongo puede efectuarse durante las primeras 24 horas de contacto con las hojas por procesos de adhesión y germinación (Cristancho et al. 2012), por lo tanto, al aplicar los tratamientos ya había empezado el proceso de infección. En el segundo tratamiento, cuando se aplican las soluciones de manera preventiva, se consiguió un excelente resultado con las 3 soluciones, lo cual sugiere que la aplicación de los productos genera un capa protectora sobre la hoja que bloquea la adhesión de las esporas e inhibe la germinación y por tanto evita el proceso de infección. Este resultado representa una herramienta de fácil acceso para ser implementada en los programas de manejo integrado, a la vez que se desarrollan especies más resistentes al patógeno y se investiga las características genéticas de la enfermedad (Toniutti et al. 2017; Silva et al. 2018). Finalmente, en el tercer tratamiento, cuando se aplican las soluciones al mismo tiempo que el inóculo, se observa una inhibición de la germinación similar al tratamiento in vitro, lo que se traduce en porcentajes de daño cercanos a 0% registrados para OQ y la mezcla ya sea por inhibición de la germinación de las esporas y/o por bloqueo de la adhesión de éstas en tejido vegetal. Este efecto de los oligómeros de quitosano fue reportado en ensayos similares contra patógenos de fresas (Rahman et al. 2015). Aunque con la aplicación de EEP la efectividad se redujo, su aplicación es efectiva respecto al control. Artículo 5. Los recubrimientos de semillas a base de quitosano (de diferente PM) y propóleo, fueron aplicados para comprobar si estos productos bioactivos eran capaces de conferir resistencia contra F. circinatum a dos especies de pinos (P. sylvestris y P. radiata). Aunque no se encontró un efecto positivo en la germinación como se ha observado en otras plantas (Zeng et al. 2012; Peña-Datoli et al. 2016), los recubrimientos de quitosano y propóleo redujeron significativamente la mortalidad de las plántulas, principalmente en P. sylvestris, con tasas de sobrevivencia superiores al 50%. Demostrado también en los análisis del TPC y RSA, pues se ha reportado que el quitosano y el propóleo tienen la capacidad de inducir mecanismos de defensa a las plantas, como la producción de fenoles y compuestos antioxidantes (Liu et al. 2007; Badawy & Rabea 2009). Es este estudio, como recubrimientos | Discusión General Biocomposites antifúngicos de base quitosano para la protección de especies agrícolas, forestales y de madera | 37 de semillas, no se observó que el quitosano de bajo PM tuviera mejores efectos que el quitosano de medio PM, como se sugiere en la mayoría de las aplicaciones (Kananont et al. 2010), excepto en el análisis de TPC, donde los OQ presentaron mejores resultados que el QMPM. Todo lo anterior sugiere que los recubrimientos propuestos son prometedores para la protección de plántulas de P. sylvestris contra PPC. Artículo 6. La actividad del QMPM, los OQ, y los composites OQ-EEP y OQ-EEP-nAg, se evaluó en la protección superficial de madera, en una prueba rápida de degradación llevada a cabo durante 30 días de exposición al hongo T. versicolor, el hongo causante de la pudrición blanca. Esta prueba, mostró una tasa de degradación mayor en Populus euroamericana que en Fagus sylvatica expuesta durante el mismo tiempo al ataque del hongo, lo que denota una mayor susceptibilidad como lo reportan Jia-qi et al. (2005) y Spavento (2015). Por otra parte, la mejor actividad protectora del quitosano de mayor PM frente al de menor y sus composites, coincide con otros estudios sobre madera de P. sylvestris (Eikenes et al. 2005), aunque evidentemente este efecto es altamente dependiente de la concentración del producto aplicado (El-Gamal et al. 2016). Respecto a la desintegración de la madera causada por el hongo monitorizada mediante microscopía y espectroscopía vibracional, se evidencian las limitaciones de los recubrimientos a base de OQ en comparación con MMWC, que tiene una mayor viscosidad y mejores propiedades de adhesión. Por tanto el uso de MMWC es prometedor para la protección de la madera de chopo, con posibles aplicaciones industriales. 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Food Chem 190:1174–1181. doi: 10.1016/j.foodchem.2015.06.076 Biocomposites antifúngicos de base quitosano para la protección de especies agrícolas, forestales y de madera | 53 Artículos originales Eco-friendly nanocomposites of chitosan 6 Chitosan / nanosilica Multiple applications (Podust et al., 2014) Chitosan / gelatin / essential oils Antimicrobial (Gómez-Estaca et al., 2010) Chitosan / gelatin Food packaging (Palma et al., 2016) Ternary (or more) Natural extracts Chitosan films / lauric arginate / cinnamon oil / ethylenediaminetetra acetate Antimicrobial (Ma et al., 2016) Natural extracts / Synthetic Chitosan / collagen / bioactive glass NPs Biomedical (Moreira et al., 2016) Natural extracts / Metals Chitosan oligomers / propolis / silver NPs Fungicide (Matei et al., 2015) Chitosan / neem oil / zinc oxide Fungicide (Sanuja et al., 2015) Synthetic Chitosan / tween 20 / span 60 Fungicide (Ziani et al., 2009) Synthetic / Metals Chitosan / glutaraldehyde / copper Biomedical (Xue & Wilson, 2016) 3.3.1. Chitosan combined with biomaterials Composites based on chitosan binary mixtures encompass the addition of species of biological 185 nature and of synthetic chemistry species in order to obtain the desired composite design. For the food industry, biopolymer-coated liposomes by electrostatic adsorption of chitosan have been prepared. It was demonstrated by fluorescence polarization that chitosan can flatly adsorb onto the membrane surface through electrostatic attraction, inducing charge inversion but maintaining the spherical shape of liposomes. These results may contribute to the development of chitosomes as 190 potential candidates for an efficient delivery of bioactive compounds in nutraceutical and functional foods (Tan et al., 2016). A biosensor based on chitosan for pesticides detection with enhanced solvent resistance was recently manufactured from the acetycholinesterase enzyme. Solvent tolerance of immobilized enzymes is important for many biosensing and biotechnological applications, and these findings can 195 enable future selection of the immobilization matrix and solvent type for the development of organic phase enzyme-based systems (Warner & Andreescu, 2016). 3.3.2. Chitosan cross-linked with natural extracts In recent years, the unparalleled and functional properties of essential oils have been widely 200 studied, but their sensitivity to environmental factors and their poor aqueous solubility have limited their applications in industries. Carum copticum essential oil was combined with chitosan nanoparticles by an emulsion ionic gelation, using pantasodium tripolyphosphate and sodium hexametaphosphte as cross-linkers. The biological properties of Carum copticum essential oil, before and after the encapsulation process, 205 were determined by FTIR and thermal analysis, concluding that the essential oil had been encapsulated into the chitosan nanoparticles without any chemical reaction. The structure and function of oil were not changed in this process, suggesting maintenance of its antibacterial and antioxidant properties (Esmaeili & Asgari, 2015). Thyme oil has also been mixed with chitosan in order to make biofilms for wound healing 210 applications, due to their antimicrobial and antioxidant activities. The results confirmed the good potential of thyme oil to be incorporated into these antibacterial and permeable films, and vibrational Eco-friendly nanocomposites of chitosan 7 spectroscopy data showed that there was no interaction between the functional groups of chitosan and the active groups of thyme oil (Altiok et al., 2010). In the same way, cinnamon essential oil was mixed with chitosan in order to make biofilms with 215 lower moisture content than pure chitosan films, which also showed improved antimicrobial activity, solubility in water, water vapor permeability and elongation at break properties (Ojagh et al., 2010). Another interesting natural extract is 4-hydroxy-3-methoxybenzaldehyde (vanillin), the primary component of the extract of the vanilla bean. Films containing chitosan and vanillin were obtained and could be used as antimicrobial and as flavor-release materials. The release kinetics of vanillin 220 from these films were evaluated and the emulsifier influence on the flavor release was also assessed (Stroescu et al., 2015). Moreover, chitosan derivatives such as N-vanillyl chitosan and 4hydroxybenzyl chitosan have also been prepared by reacting the amino groups of chitosan with these aldehydes to form a Schiff base intermediate, which was subsequently converted into N-alkyl chitosan by reduction with sodium cyanoborohydride (Figure 3.4). The antimicrobial activity of the 225 modified chitosan films was studied against Aspergillus flavus, finding a clear reduction of the aflatoxins produced by the fungus (to 98.9% and non-detectable levels, for N-vanillyl chitosan and 4-hydroxybenzyl chitosan film discs, respectively) (Jagadish et al., 2012). 230 Figure 3.4. Synthesis of N-vanillyl chitosan, as an intermediate step towards the obtaining of N-alkyl chitosan films. 235 The effectiveness of chitosan coated polypropylene films against bacteria, either in its pure form or incorporating an ethanolic extract of propolis (EPP), has also been evaluated against six foodborne pathogens (Salmonella typhimurium, Escherichia coli, Staphylococcus aureus, Listeria monocytogenes, Cronobacter sakazakii and Bacillus cereus), demonstrating that the chitosan-coated films had a broad-spectrum antibacterial activity. However, the mechanical properties of chitosan– 240 propolis coated films are still subject of further research (Torlak & Sert, 2013). Likewise, a good antimicrobial activity was also exhibited by three formulations of propolis-based chitosan varnish, obtained by dissolution of propolis with chitosan in an hydro-alcoholic vehicle at different concentrations (Franca et al., 2014). Matei et al. (2015) conducted the synthesis of chitosan oligomers/propolis/silver nanoparticles 245 composite systems and studied their activity against a xylophagous fungus (Diplodia seriata). In this case, the vibrational spectra suggested the existence of hydrogen bonding between chitosan and propolis. 250 Eco-friendly nanocomposites of chitosan 8 3.3.3. Chitosan co-polymerized with synthetic species The nature of chitosan allows the incorporation of synthetic materials in its polymeric structure. In this way, a chitosan–chloroquinoline derivative was prepared by a Green-Chemistry technique, treating a chitosan solution in aqueous acetic acid with a 2-chloroquinoline-3-carbaldehyde solution to form a hydrogel, which was then subjected to solvent exchange. The antimicrobial activity of the 255 derivative against different bacteria, viz. Staphylococcus aureus, Escherichia coli and Candida albicans was evaluated, concluding that the chitosan–chloroquinoline derivative holds promise for novel antimicrobial agents (Kumar et al., 2011). Tahtat et al. (2011) investigated the antibacterial activity of polyvinyl alcohol hydrogels containing different concentrations of chitosan, cross-linked by γ-irradiation, against Escherichia coli 260 and Bacillus subtilis, revealing a higher effectiveness in the inhibition of the growth of gram positive bacteria than for gram negative ones. They also found that both the chitosan content in the hydrogel and its MW had a direct influence on growth inhibition. A bioactive coating consisting of a silica xerogel/chitosan hybrid -more hydrophilic with increasing silica xerogel contentwas applied by Jun et al. (2010) to titanium at room temperature as 265 a novel surface treatment for metallic implants. This hybrid coating layer induced the rapid precipitation of apatite on its surface when immersed in a simulated body fluid, thus affording excellent bone bioactivity. In comparison to a pure chitosan coating, the hybrid coating enhanced the viability of the cultured osteoblastic cells and promoted the alkaline phosphate activity of the cells, with optimum results for the composite containing 30% chitosan. Consequently, these silica 270 xerogel/chitosan hybrids can potentially be utilized as room temperature bioactive coating materials on titanium-based implants. Chitosan and bilayered – Rhodamine 6G impregnated silica–chitosan – coatings have also been prepared and investigated as a model for controlled drug release. Spectroscopic ellipsometry measurements showed that covalent cross-linking led to an increased swelling degree of chitosan 275 layers. Despite the swelling behavior, both cross-linked chitosan layers showed significant retard effect on dye release from the bilayered coatings (Dabóczi et al., 2016). Novel organic–inorganic hybrid materials consisting of chitosan and nanosilicas (plain silica, silica-titania and silica-alumina) were prepared by Podust et al. (2014) and assessed as adsorbents of biomedical relevance. The chitosan modified nano-oxides were obtained by equilibrium adsorption 280 method. It was found that chitosan adsorption capacities of TiO2/SiO2 and Al2O3/SiO2 were higher than that of the plain SiO2 because the mixed oxides presented additional active sites on their surfaces. Other research groups have conducted studies by combining chitosan with oils and/or plasticizers, to yield different types of composites or nanocomposites, such as edible films consisting of gelatin-chitosan incorporated with clove essential oil, whose antimicrobial activity was tested 285 against six selected microorganisms (namely Escherichia coli, Listeria innocua, Pseudomonas fluorescens, Shewanella putrefaciens, Lactobacillus acidophilus and Photobacterium phosphoreum). The gelatin-chitosan film incorporating clove essential oil was then applied to fish during chilled storage, observing a significant reduction in the growth of gram-negative bacteria, in particular Enterobacteriaceae, while the impact on lactic acid bacteria was negligible. The addition of clove 290 essential oil to the film also led to a substantial increase in water solubility, which can be ascribed to protein–polyphenol interactions that weaken the interactions that stabilize the protein net (GómezEstaca et al., 2010). The mechanical properties of the chitosan–gelatin edible films were assayed and the interaction between gelatin, chitosan and several plasticizers, pure or in binary combinations, was also investigated (Palma et al., 2016). 295 Furthermore, chitosan films with lauric arginate, cinnamon oil, and ethylenediaminetetraacetate (EDTA) have been prepared, and their combination results in a synergistic antimicrobial effect on gram-positive bacteria but with an antagonistic effect on gram-negative bacteria (Ma et al., 2016). Eco-friendly nanocomposites of chitosan 9 Stimuli-responsive nanocomposite-derived hydrogels have recently gained prominence in tissue engineering because they can be applied as injectable scaffolds in bone and cartilage repair. Due to 300 the great potential of these systems, Moreira et al. (2016) synthesized and characterized novel thermosensitive chitosan-based composites, chemically modified with collagen and reinforced by bioactive glass nanoparticles, aimed at the development of injectable nanohybrids for regenerative medicine applications. The results demonstrated that the addition of collagen and bioactive glass increased the mechanical properties after the gelation process, with promising potential to be used as 305 thermoresponsive biomaterials for biomedical applications including bone tissue regeneration. Bionanocomposite film with enhanced properties were prepared by Sanuja et al. (2015) by incorporating different concentrations (0.1, 0.3 and 0.5%) of nano-ZnO and neem essential oil into chitosan by a solution cast method. The results showed that the 0.5% nano-ZnO incorporated composite film had improved tensile strength, film thickness, film transparency elongation, and 310 decreased water solubility, swelling and barrier properties. As mentioned above, the need to reduce the negative impact of conventional treatments on human health and on the environment has led to an increase in use of eco-friendly polymers as antimicrobial materials. The antifungal properties of films based on chitosan have been assessed in various studies, such as the work by Ziani et al. (2009), who tested its antifungal activity against 315 Aspergillus niger, Alternaria alternata and Rhizopus oryzae, showeing that the antifungal activity largely depended on the particular type of fungus treated. A one-pot kinetic uptake study of urea in aqueous solution with various chitosan sorbent materials such as pristine chitosan, cross-linked chitosan with glutaraldehyde from low to higher glutaraldehyde content, and a Cu(II) complex of a glutaraldehyde cross-linked chitosan material was 320 recently studied by Xue and Wilson (2016). Cross-linked chitosan displayed relatively rapid urea uptake and greater adsorption capacity when compared with pristine chitosan. These results further illustrate the rational design of chitosan-based materials for the controlled uptake of urea in aquatic environments. 325 3.4. Synthesis methods for chitosan-based nanocomposites Different chitosan products have different structures and physicochemical properties, that may result in novel bioactivities or new findings in known bioactive compounds (Zou et al., 2016). As noted above, compared to the other natural polysaccharide polymers, chitosan also suffers 330 from some disadvantages, one being its low water solubility at physiological pH. The transfection efficiency of native chitosan is also relatively low, and it lacks some functionalities that are highly desired for some applications. Thus, a number of chemical modification techniques have been used to overcome these drawbacks (Taşkın et al., 2014). Chemical modification of chitosan can be attained by N-substitution, by O-substitution, or by N,O-substitution, and also via chitosan association with 335 small molecules or macromolecules. The antimicrobial activity can be improved by chemical modifications of the amino group at the C-2 position of glucosamine with positively charged groups Figure 3.5(a) (Jeon & Kim, 2001). Chitosan acylation is a significant functionalization method used to prepare chitosan derivatives with good water solubility, biocompatibility, increased bioactivities and even improved antioxidant 340 activity. A remarkable member of N-acyl chitosan group that exhibits good compatibility is Nsuccinyl-chitosan. This compound (see Figure 3.5(b)), obtained through a simple reaction between chitosan and succinic anhydride, preserves a series of biological properties such as biocompatibility and nontoxicity. Mercapto chitosan can be obtained by thioglycolic acid (SHCH2COOH) thiolation reaction (Figure 3.5(c)). Chemical modification of chitosan by acylation and copolymerization are 345 shown in Figure 3.5(d) and Figure 3.5(e), respectively. Eco-friendly nanocomposites of chitosan 10 Figure 3.5. Synthetic methods for the chemical modification of chitosan. 350 Eco-friendly nanocomposites of chitosan 11 Table 3.2. Synthesis methods for the obtaining of composites based on chitosan. Type of synthesis Synthetic methods Materials Applications References Biological Biocatalysis with microbial transglutaminase Chitosan /nisin Antimicrobial (Zhu et al., 2015) Enzymatic hydrolysis Chitosan Multiple applications (Pan et al., 2016) Physical Ultrasonication Chitosan Food packaging (Ho et al., 2016) Thermal treatment Chitosan / nanosilver Antimicrobial (Wei et al., 2009) Microwave Chitosan / TETA Environmental (Ge & Ma, 2015) Ultrasonication Chitosan NPs Multiple applications (Gokce et al., 2014) Chemical Graft polymerization (ATRP) Chitosan / lactic acid Multiple applications (Bhattarai et al., 2006) Catalysis Chitosan / nanosilver Antimicrobial (Venkatesham et al., 2012) Acid hydrolysis Chitosan acrylic NPs Antimicrobial (Torabi et al., 2016) Chemical coupling Chitosan / nanosilver Antimicrobial (Gu et al., 2014) Acid hydrolysis / catalysis Chitosan Multiple applications (Xia et al., 2013) Sol–gel method by freeze casting Chitosan / bioactive glass Biomedical (Pourhaghgouy et al., 2016) Nucleophilic substitution Chitosan / pyridine Fungicide (Jia et al., 2016) 355 3.4.1. Biological methods The modification of chitosan with enzymes, in comparison to chemical modification, can be regarded as particularly attractive because of its specificity and also in terms of its environmental impact (Shukla et al., 2013). Among the methods using the enzymatic pathway for obtaining chitosan 360 composites we have selected, based on their advocated effectiveness, one that uses microbial transglutaminase and nisin natural antimicrobial and another that employs papain (Table 3.2). By using microbial transglutaminase as a biocatalyst, nisin grafted chitosan was prepared with high selectivity and efficiency, under mild reaction conditions and in an environmental friendly way. By adjusting the reaction temperature, the reaction time and the molar ratio of nisin to chitosan, it 365 was possible to control the degree of substitution of nisin–chitosan, and the resulting product showed excellent solubility at different pH. This composite can find application in, for example, the pharmaceutical and food industry fields (Zhu et al., 2015). Pan et al. (2016) obtained low molecular weight chitosan by the enzymolysis of chitosan by papain. Enzymolysis conditions, the initial chitosan concentration, temperature, pH and ratio of 370 papain to chitosan were optimized by conducting experiments at three different levels using the response surface methodology to obtain high soluble reducing sugars concentrations. Meanwhile, the influence of chitosan substrate concentration on the activity of papain was assessed and chitosan exhibited substrate inhibition. 375 3.4.2. Physical methods The physical modification of chitosan can be attained by blending, or by physically mixing, at least two polymers to create a new material with different physical properties. Four examples of synthesis methods that make use of ultrasonic pretreatment, heat treatment, microwave irradiation or ionic gelation with ultrasonication techniques are discussed below. 380 Eco-friendly nanocomposites of chitosan 12 Self-aggregated chitosan particles are a promising candidate in stabilizing food-based emulsions because they are naturally-derived, edible, and inexpensive. In a recent study, the self-aggregated chitosan particles were synthesized from chitosan solution with and without ultrasonication pretreatment. Ultrasonication pretreatment caused depolymerization of chitosan, resulting in the formation of smaller and monodisperse chitosan particles, in comparison to the non-pretreated 385 chitosan. These findings suggest that ultrasonication pretreatment on chitosan could reduce the hydrophobicity of the chitosan particles formed via self-aggregation, as confirmed by contact angle measurements (Ho et al., 2016). Wei et al. (2009) synthesized chitosan-based Ag-NPs by reducing AgNO3 with chitosan. The resulting Ag-NPs exhibited a high antibacterial activity toward both gram-positive and gram-negative 390 bacteria, comparable with that of the highly active silver salts precursor. The Ag-NPs impregnated chitosan films, formed via thermal treatment, showed both faster and more long-lasting antibacterial effectiveness against Escherichia coli than pure chitosan films. A novel triethylenetetramine/graphene oxide/chitosan composite was successfully synthesized by microwave irradiation method and compared with one prepared by conventional heating. The 395 experimental results indicated that the product obtained using microwaves had higher yield and uptake than the one obtained by the conventional approach (Ge & Ma, 2015). An ionic gelation method have also been used for the preparation of chitosan nanoparticles. The impact of freeze-drying, ultrasonication time and cryoprotectant (d-trehalose) utilization on the particle size, size distribution and stability of the nanoparticles as evaluated, concluding that freeze400 drying caused the particles to agglomerate, that the addition of the cryoprotectant led to a decrease in the particle size and increasing ultrasonication time increased the nanoparticles size. Accordingly, Gokce et al. (2014) determined that controlled ultrasonic treatment and the use of cryoprotectant were effective for imparting nanoparticle stability. 405 3.4.3. Chemical methods Physicochemical properties, such as electrostatic charging and permeation of polymeric surfaces, can be altered by chemical modification, which can take place in different ways. Amongst the many chemical modifications available, alkylation, acylation, hydroxylation, nitration, sulphonation, phosphorylation, xanthation, Schiff’s base formation and graft copolymerization are 410 the most popular approaches (Shukla et al., 2013). Graft co-polymerization can be defined a method in which one polymer is covalently bonded to the other polymer chain (Benamer et al., 2011). Among the various graft copolymerization techniques used to change the surface characteristics of polymers such as chitosan and other materials for different applications, the technique of atom transfer radical polymerization (ATRP) is rapidly 415 emerging as the first preference due to the advantages it offers over other techniques (Ifuku et al., 2013; Thakur & Thakur, 2014). Bhattarai et al. (2006) attained lactic acid modification of chitosan without using a catalyst by grafting D,L-lactic acid onto amino groups in chitosan. The following are some representative examples of chemical synthesis methods that incorporate catalysis, acid hydrolysis, chemical coupling, sol-gel method and nucleophilic substitution procedures 420 for the obtaining of composites based on chitosan. Stable silver nanoparticles were synthesized by Venkatesham et al. (2012), without using any toxic chemicals, by using chitosan as both a reducing and a stabilizing agent. The antimicrobial activity of the resulting Ag-NPs was tested against Micrococcus luteus and Escherichia coli, confirming inhibiting properties. 425 Torabi et al. (2016) also reported an environmentally-friendly process for the preparation of acrylic/chitosan films with antibacterial activity and nontoxic properties by using a water-base acrylic Eco-friendly nanocomposites of chitosan 13 resin. It was found that the obtained films had enhanced antibacterial activity against Escherichia coli and Staphylococcus areus, and the cytotoxicity analysis showed a reasonably non-toxic behavior of the composite films. 430 Amphiphilic chitosan-graft-poly(ε-caprolactone) copolymers were synthesized by a homogeneous coupling method and characterized by ninhydrin assay and NMR and FTIR spectroscopies. The graft copolymers were subsequently self-assembled into micelles, which were measured by dynamic light scattering and transmission electronic microscopy. The particle size of the micelles decreased as the segment grafting fraction was increased. Thereafter, silver nanoparticles 435 were prepared in the presence of chitosan-based micelles under ultraviolet irradiation. The films impregnated with the Ag-NPs showed a strong antimicrobial activity against Escherichia coli and Staphylococcus aureus (Gu et al., 2014). In the study by Xia et al. (2013), water soluble chitosan was prepared by hydrolyzation using H2O2 and phosphotungstic acid (PTA, H3PW12O40) as a catalysis in homogeneous phase, and the 440 various factors affecting hydrolysis and the optimal hydrolysis conditions were investigated. The resulting products were composed of chitooligosaccharides. A recent study by Pourhaghgouy et al. (2016) focused on chitosan-based nanocomposite scaffolds preparation by freeze-casting method through blending of a constant chitosan concentration with different portions of synthesized bioactive glass nanoparticles. The biodegradation study showed 445 that increase in bioactive glass nanoparticles content led to growth of weight loss amount, while the in vitro biomineralization studies confirmed the bioactive nature of all nanocomposites. Jia et al. (2016) introduced pyridine moieties into chitosan by nucleophilic substitution to afford N-(1-carboxybutyl-4-pyridinium) chitosan chloride (pyridine chitosan). The antifungal activity of the resulting chitosan derivative was then examined by studying the inhibition of mycelia growth and 450 spore germination. The results indicated that pyridine chitosan exhibited enhanced antifungal activity by comparison with pristine chitosan. Non-toxicity of pyridine chitosan was demonstrated by an acute toxicity study. These results are beneficial for assessing the potential utilization of this chitosan derivative and for exploring new functional antifungal agents with chitosan in the food industry. 455 3.5. Analytical techniques for the identification of the composite materials The identification of characteristics such as the degree of deacetylation, MW, polydispersity and crystallinity are of utmost importance for the synthesis of chitosan derivatives. For instance, amongst the different analytical techniques available, atomic force microscopy (AFM), cryogenic transmission 460 electron microscopy (cryo-TEM) and small-angle neutron scattering (SANS) analysis have been used to characterize the molecular shape and water soluble grafted chitosan (see Table 3.3) (Shukla et al., 2013). 465 Eco-friendly nanocomposites of chitosan 14 Table 3.3. Methods for the determination of the physicochemical characteristics of chitosan and chitosan derivatives. Updated from that available in (Trutnau et al., 2011). Physico-chemical characteristics Determinations methods Reference Degree of deacetylation Infrared spectroscopy (FT-IR) (Martínez-Camacho et al., 2010) First derivative UV-spectroscopy (Kasaai, 2009) Nuclear magnetic resonance spectroscopy (NMR) (Aranaz et al., 2009) Titration (alkalimetric, conductometric, potentiometric) (Trutnau et al., 2009) Differential scanning calorimetry (DSC) (Jiang et al., 2003) Molecular weight /Mw distribution Viscosimetry (Rhazi et al., 2000) Gel permeation chromatography (Brugnerotto et al., 2001) Light scanning (Chatterjee et al., 2005) Electrophoresis (Muzzarelli et al., 1994) Crystallinity X-ray diffraction (XRD) (Matei et al., 2015) Moisture content Gravimetric analysis (Ogawa et al., 1993) Ash content Gravimetric analysis (Ogawa et al., 1993) Protein content Bradford method (Dornish et al., 2001) Texture Transmission electronic microscope (TEM) (Pillai et al., 2009) Morphological structures Scanning electronic microscope (SEM) (Sanuja et al., 2015) Thermal stability Thermo gravimetric analysis (TGA) (Martínez-Camacho et al., 2010) Mechanical properties Near-infrared (Palma et al., 2016) 470 3.6. Advanced applications of bionanomaterials based on chitosan In this section some of the applications of the nanocomposites are discussed: as antimicrobial agents for the control of fungi and bacteria; in biomedical applications for wound dressings, drug delivery or tissue engineering; in food packaging; in environmental applications for the adsorption of metals and toxics or in wastewater treatment; in crop protection; and in the conservation cultural of 475 heritage such as historic and artistic works in stone, wood, textile, etc. (Figure 3.6). Eco-friendly nanocomposites of chitosan 15 Figure 3.6. Chitosan applications in different fields. 480 3.6.1. Antimicrobial applications Compounds of chitosan are being widely researched for their application as antimicrobial agents. Ziani et al., 2009 examined the effect of chitosan with different MWs at different concentrations as 485 an antifungal in the control of phytopathogenic fungi Aspergillus niger, Alternaria alternata and Rhizopus oryzae. The highest inhibition was observed against A. alternata (97%). As noted above, a chitosan-nisin composite also showed a marked inhibitory effect against Bacillus subtilis, Escherichia coli and Staphylococcus aureus, which is very promising for both pharmaceutical and food-related uses (Zhu et al., 2015). On the other hand, Torabi et al. (2016) 490 illustrated that a blend of polyacrylic nanoparticles with chitosan is an easy and efficient method for obtaining anti-bacterial coatings, non-toxic and with a good control of the desired properties. Several recent studies also demonstrate the antibacterial activity of other composites with chitosan (Esmaeili & Asgari, 2015; Gómez-Estaca et al., 2010; Gu et al., 2014; Ma et al., 2016; Tahtat et al., 2011; Torlak & Sert, 2013; Venkatesham et al., 2012). These compounds were tested for use 495 as bactericides in food packaging, in the pharmaceutical, nutraceutical and cosmetic industries, confirming their inhibitory effect against Escherichia coli, Staphylococcus aureus, Bacillus cereus, Bacillus subtilis, Cronobacter sakazakii, Listeria monocytogenes and Salmonella typhimurium. A compound of chitosan and antifungal pyridine was tested against Fulvia fulva and Botrytis cinerea, resulting in a superior antifungal activity compared with pure chitosan. This nontoxic 500 compound can be deemed as very promising as a new antifungal agent in the food industry (Jia et al., 2016). Matei et al. 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Revista: Journal of Spectroscopy Editorial: Hindawi Publishing Corporation Año: 2016 Páginas: 1-7 DOI: http://dx.doi.org/10.1155/2016/4058478 1 A study of the far infrared spectrum of N-acetyl-D-glucosamine using THz-TDS, FTIR and semi-empirical quantum chemistry methods Pedro Chamorro-Posada1, Iosody Silva-Castro2, José Vázquez-Cabo3, Pablo Martín-Ramos4, José 5 María López-Santos3 and Jesús Martín-Gil2* 1 Dpto. de Teoría de la Señal y Comunicaciones e IT, Universidad de Valladolid, ETSI Telecomunicación, Paseo Belén 15, 47011 Valladolid, Spain. 2 Advanced Materials Laboratory, ETSIIAA, Universidad de Valladolid, Avenida de Madrid 44, 34004 10 Palencia, Spain. Tel: +34 (979) 108347; Fax: +34 (979) 108301; e-mail: [email protected]. 3 Dpto. de Teoría de la Señal y Comunicaciones, Universidad de Vigo, ETSI Telecomunicación, Lagoas Marcosende s/n, Vigo, Spain. 4 EPSH, Universidad de Zaragoza, Carretera de Cuarte s/n, 22071 Huesca, Spain. 15 Abstract The far-infrared spectrum of N-acetylglucosamine has been studied by combining THz–TDS and FT-IR characterization techniques with theoretical studies based on semi-empirical quantum chemistry methods. A strong spectral peak at 1.8 THz has been identified, which constitutes the 20 main signature of the material in the terahertz band. Calculated molecular vibrations are in good qualitative and semi-quantitative agreement with both the THz-TDS and FT-IR experiments. In comparison to previous DFT-based studies, the semiempirical approach chosen herein, suitable for parallel multi-core and GPU acceleration, allows for a full study using periodic boundary conditions and no further approximations within a constrained computing time. 25 Keywords: FIR spectroscopy; FTIR spectroscopy; N-acetylglucosamine; THz-TDS spectroscopy; semiempirical methods. 30 1. Introduction N-acetylglucosamine (or D-GlcNAc, or NAG) is a derivitized glucose monomer found in polymers of bacterial cell walls, chitin, hyaluronic acids and various glycans. As the monomeric unit of chitin polymer, it forms the outer coverings of insects and marine crustaceans and it is the major component of the cell walls of most fungi. Chitosan (deacetylchitin) is a form of N35 acetylglucosamine that has been chemically altered. N-acetylglucosamine derivatives -chitin and chitosanhave become of great interest not only as underutilized resources, but also as new functional materials of high potential in various fields [1-3]: in biomedical applications (antimicrobial agents, drug and gene delivery, wound dressings and tissue engineering), in waste water treatment (purification and toxic ion removal), agriculture 40 (seed coatings and controlled agrochemical release), in food industry (packaging and preservative materials) and in cosmetics, to name a few. The structure of N-acetylglucosamine [4] is shown in Figure 1. Crystals are monoclinic, with space group P21 and unit cell parameters a=11.25 Å, b=4.82 Å, c=9.72Å, and β=113.7o. Mercury software package [5] has been used for the representation. 45 2 Figure 1. Crystal cell of N-acetyl-D-glucosamine. 50 The far infrared (FIR) spectra of polycrystalline monoand poly-saccharides is dominated by lattice modes involving molecular hydrogen bonds or/and van der Waals forces. Therefore, FIR spectroscopy techniques in general and, particularly, terahertz time-domain spectroscopy (THzTDS) are notably useful in the structural characterization of carbohydrates [6-8]. Computational quantum chemistry is a highly valuable tool for the interpretation of FIR spectra 55 due to the complexity of the associated vibration modes. Ab-initio and density functional theory (DFT) methods are very accurate in the prediction of molecular vibrations [9]. Nevertheless, solidstate calculations over large supra-molecular systems are typically required due to the relevance of long-range interactions, thus limiting the use of these computationally costly methods. On the other hand, semi-empirical methods [10,11], especially with the recently developed parallel 60 implementations for shared-memory multiprocessor and massively-parallel graphics processing units architectures [12], permit to study the vibrations in large molecular crystals at reduced computation times. Semi-empirical quantum chemistry methods have been previously used in the interpretation of the terahertz spectra of various carbon nitride [13] and carbon [14] materials. A study of the IR spectrum of NAG was previously presented in [15]. The experimental 65 characterization was performed using a FT-IR spectrometer and the theoretical modeling using a DFT method. Due to the high computer cost of such calculations, a single molecule was studied and the intermolecular hydrogen bond interactions were simulated with intramolecular interactions. In this work, we focus on the far-infrared spectrum of NAG. We combine THz–TDS and FT-IR techniques for the characterization of the spectral regions between 10 cm-1 and 80 cm70 1 and 80 cm-1 and 420 cm-1, respectively. THz-TDS measurements have permitted to identify one strong spectral peak at 1.8 THz, which is the main signature of the material in the terahertz band. Theoretical studies have been performed using semi-empirical quantum chemistry methods. The use of parallel multi-core and GPU acceleration allows for a full study using periodic boundary conditions and no further approximations within a constrained computing time. 75 2. Materials, experimental and theoretical methods 2.1. Materials N-acetyl-D-glucosamine (CAS No. 7512-17-6, ≥99%) was purchased from Sigma-Aldrich 80 Chemie GmbH (Schnelldorf, Germany). The sample material was mixed with ultra-high molecular weight surface-modified, 53-75 µm particle size polyethylene (PE, CAS No. 9002-88-4), also from Sigma-Aldrich, at 18 wt%. The mixture was pressed at 5 tm for 3 minutes to make a 13 mmdiameter pellet. 85 3 2.2. Spectroscopic characterization A Menlo (Martinsried, Germany) Tera K15 spectrometer was used for the THz-TDS analysis. The system is based on a 1560 nm fiber laser that generates 90 fs pulses at a repetition rate of 100 MHz. This provides a compact fiber-coupled setup. The system was operated in a nitrogen rich 90 atmosphere in order to avoid the signature of water absorption in the recorded samples. Ten sample and ten reference measurements were performed in each case in order to reduce the noise in the measurements. The material parameters in the spectral range of interest were calculated from the time domain photocurrent traces measured with the spectrometer. These time domain waveforms depend not 95 only on the material data but also on the width of the pellets, due to the contributions from multiple reflections at the pellet-air interfaces. Signal processing techniques similar to those described by Duvillaret et al. [16], with a flat-top window [17], were employed in order to obtain the THz spectra of the materials. The vibrational spectrum of the material in the 80-420 cm-1 spectral range was measured using 100 a Thermo Scientific (Waltham, MA, USA) Nicolet iS50 FT-IR Spectrometer. At shorter wavelengths the measurement was saturated because of the attenuation associated to the PE matrix. 2.3. Quantum chemistry computations 105 The semiempirical quantum chemistry computations were performed with the PM6 method [18] using the parallel implementation for multi-threaded shared-memory CPUs and massively parallel GPU acceleration [12] of the MOPAC2012 [19] software package. A Fedora Linux server with a 12-core Intel Xeon processor and a NVIDIA Tesla K20 GPU were used for the computations. 110 3. Results and discussion 3.1. FIR spectrum of N-acetyl-D-glucosamine The results of the THz-TDS measurements are shown in Figure 2. The terahertz spectrum in 115 the spectral range from 0.3 THz to 2.5 THz (80 cm-1) was characterized by a dominant peak at 1.8 THz. Two very weak peaks could also be observed at 1.36 THz and 2.15 THz, respectively. Figure 2. N-acetyl-D-glucosamine THz-TDS measurement results. 120 4 The FT-IR measurement results in the spectral range from 80 cm-1 to 420 cm-1 are shown in 125 Figure 3. The solid line corresponds to the sample material dispersed in PE powder and the dashed line to a pure polyethylene reference pellet. The polyethylene absorption displayed the expected Rayleigh scattering monotonic increase of attenuation with frequency associated with the finite particle size, and the PE band at 110 cm-1 [20]. A second faint PE absorption band was also found close to 230 cm-1. The spectrum in the whole range is shown in Figure 4. 130 Figure 3. FT-IR spectrum in the spectral range from 80 cm-1 to 420 cm-1. The solid line corresponds to Nacetyl-D-glucosamine dispersed in polyethylene powder at 18 wt% and the dashed line to a reference pellet of pure polyethylene. 135 Figure 4. Measured FT-IR spectrum (dashed blue line), after subtraction fo the contribution to the absorbance of the PE matrix, and THz-TDS measurement (red solid line). 140 3.2. Semi-empirical quantum chemistry results The solid-state geometry of NAG was optimized using the PM6 Hamiltonian [18] using the actual crystal geometry [4] as the initial condition. Accurate calculations using MOPAC with periodic boundary conditions require a computational domain at least capable of fitting a sphere 145 with a diameter of 8 Å. A minimal domain spanning 1×2×1 crystal unit cells was used by setting the keyword MERS=(1,2,1). The unit cell dimensions of the resulting optimized geometry a=11.400 Å, b= 4.616 Å and c=9.040 Å were in relatively good agreement with the actual values a=11.25 Å, b=4.82 Å, and c=9.72 Å. Nevertheless, the cell angles α=94.56o, β=114.09o and γ=85.51o showed a noticeable deviation from the monoclinic unit cell, even though β was very 150 close to the experimental value of 113.7o. The calculated vibrations for the crystal geometry optmized with the PM6 Hamiltonian did not include any imaginary frequency. PM7 [21] calculations provided a better fit with the actual crystal unit cell angles, but the method failed to converge to a true ground state geometry. 5 The PM6 geometry of one of the two molecules of the crystal unit cell is compared with the 155 experimental structure in Figure 5. The two molecules have been plotted using VMD [22]. A relative shift has been introduced to facilitate the visual comparison due to the highly overlapping geometries. Bond lengths are compared in Table 1, and angles in Table 2. We have used the atom labels shown in Figure 6. 160 Figure 5. Comparison of the PM6 optimized geometry with the experimental X-ray geometry of N-acetylD-glucosamine. 165 Figure 6. Atom labels used in the comparison of the molecular geometries. Table 1. Comparison of the experimental bond lengths (in Å) with those of the geometry optimized with the PM6 method. The unsigned mean error (UME) was 1.30%. 170 Bond Experimental distance PM6-calculated distance Error C1-C2 1.5281 1.5499 1.43% C2-C3 1.5367 1.5477 0.72% C3-C4 1.5344 1.5464 0.78% C4-C5 1.5504 1.5392 0.72% C5-C6 1.5201 1.5298 0.64% C7-C8 1.5126 1.4903 1.47% C2-N1 1.4763 1.4745 0.12% C7-N1 1.3800 1.3819 0.14% C1-O1 1.3935 1.4167 1.66% C3-O2 1.4511 1.4319 1.32% C4-O3 1.4685 1.4382 2.06% C1-O4 1.4593 1.4248 2.36% C5-O4 1.4598 1.4581 0.12% C6-O5 1.3908 1.4402 3.55% C7-O6 1.2177 1.2465 2.37% 1 Silver Nanoaggregates on Chitosan Functionalized Reduced Graphene Oxide using Microwaves Radiation Iosody Silva-Castro1, Pablo Martín-Ramos2*, Mercedes Sánchez-Bascones3, Luis M. Navas-Gracia1 5 and Jesús Martín-Gil1 1 Agriculture and Forestry Engineering Department, ETSIIAA, Universidad de Valladolid, Avenida de Madrid 44, 34004 Palencia, Spain. 2 Department of Agricultural and Environmental Sciences, EPSH, University of Zaragoza, Carretera de 10 Cuarte, s/n, 22071 Huesca, Spain. E-mail: [email protected]; Tel: +34 (974) 292668; Fax: +34 (974) 239302. 3 Agriculture and Forestry Science Department, ETSIIAA, Universidad de Valladolid, Avenida de Madrid 57, 34004 Palencia, Spain 15 Abstract A reduced graphene oxide (rGO)/chitosan oligomers (CSO)/silver nanoparticle (AgNPs) composite was designed and prepared via a self-assembly process with rGO through a microwave20 assisted method. The material, isolated as a nano-crystalline graphenic material, was characterized by ATR-FTIR and UV-Vis spectroscopies, X-ray powder diffraction, TEM microscopy and energy-dispersive X-ray spectroscopy. The nano-rGO/CSO/AgNPs composite can be described as a biopolymer in which AgNPs have been anchored on the surface of CSO-functionalized graphitic rGO sheets. 25 Keywords: chitosan; nanocomposite; reduced graphene oxide; self-assembly; silver nanoparticles 30 1. Introduction Mesoporous networks in graphene oxide (GO) composite structures are particularly appropriate for the formation of nanoparticles in comparison with traditional routes [1-7]. On the other hand, chitosan (CS), a well-known compound resulting from chitin N-deacetylation, shows functional amine segments (carboxyl, hydroxyl, epoxy, and –NH2 groups) in the molecular skeleton that can 35 form nanostructures with GO or reduced graphene oxide (rGO) via interactions such as hydrogen bonding. Morphological characterization of rGO/GO-CS composites have demonstrated the formation of porous 3D nanostructures which feature a high adsorption capacity. By contrast, negatively charged silver nanoparticles (AgNPs) are prone to form aggregates on GO via electrostatic interaction [8] and are susceptible to be enveloped by chitosan, CS [9]. Thus, 40 the integration of aforementioned materials in a one nanomaterial can be deemed as an achievable goal. In fact, GO/CS/AgNPs and rGO/CS/AgNPs ternary nanocomposite systems have been designed so that anchoring of AgNPs to CS functionalized graphitic GO/rGO sheets can occur [810]. The present study is focused on investigating the preparation of rGO/CSO/AgNPs 45 nanocomposites (were CSO stands for chitosan oligomers) through a microwave-assisted method, which has not been reported before to the best of the authors’ knowledge. This emerging synthesis technique has already proved to be a valuable alternative to selectively prepare materials and nanomaterials, in almost quantitative yields and with greater precision than using conventional heating [11-13]. 50 2 2. Experimental section 2.1. Materials and methods High quality reduced graphene oxide (rGO), produced from helical-ribbon carbon nanofibres by 55 chemical methods, was supplied by Grupo Antolín Ingeniería (GRAnPH Nanotech, Burgos, Spain). The sample consists of few layer (up to five) graphene oxide nanoplatelets, highly crystalline and with a large cross section (above 10 square microns) [14]. Medium molar mass chitosan (CAS No. 9012-76-4) was purchased from Hangzhou Simit Chemical Technology Co. Ltd (Hangzhou, China). Silver nitrate (CAS No. 7761-88-8) was 60 supplied by Merck Millipore (Darmstadt, Germany). The formation and assembly of rGO/CSO/AgNPs was carried out in a Milestone Ethos-One microwave. An ultrasonic machine, model CSA 20-S500, 20 kHz was used for the sonication of the solutions. Infrared spectra were recorded with a Thermo Nicolet 380 FT-IR apparatus, equipped with 65 Smart Orbit Diamond ATR system, in order to identify the chemical functional groups. Optical absorption spectra in the UV-Vis region were recorded with a Shimazdu UV-2450 UV-Vis spectrophotometer. X-ray powder diffractograms of the samples were obtained using a Bruker D8 Advance Bragg-Brentano diffractometer, in reflection geometry. Transmission electron microscope (TEM) micrographs were collected with a JEOL JEM-FS2200 HRP equipped with an Oxford 70 instruments INCA Energy TEM 250 EDS probe. 2.2. Preparation 2.2.1. Chitosan oligomers preparation 75 An aqueous solution of chitosan oligomers (CSO) was prepared from chitosan with average molecular weight (140000-300000 g/mol) in 2% AcOH at pH 4-6 with the addition of 0.3 M H2O2, under constant agitation for 12 hours. Subsequently, it was subjected to 6 intermittent periods of sonication, of 5 minutes each, keeping the temperature below 60 °C. The molecular weight of the resulting oligomer was about 2000 g/mol, in accordance with the tests reported by Sun et al. [15]. 80 2.2.2. Silver NPs preparation Silver nanoparticles were prepared by a sonication method, without resourcing to UV stabilization (used, for example, in [16]), as follows: an aqueous solution of AgNO3 (50 mM) was treated with sodium citrate (30 mM) and the resulting solution was cooled and stirred at a 85 temperature between 5 and 10 °C. Subsequently, it was deoxygenated with an inert gas (N2) for over 30 minutes and the pH was adjusted between 7 and 8. Polyvinylpyrrolidone was added to prevent the silver nanoparticles aggregation. A 10 mM solution of NaBH4 (reducing agent) was then added dropwise: the first droplet made the solution turn from colorless to yellowish and successive droplets led to an intensification of the yellow color (care had to be taken so as to avoid 90 an excess of reducing agent, which would lead to a brownish color). After vigorous stirring for one hour, the yellowish solution was sonicated for 3-5 minutes and then allowed to rest and stabilize for at least 24 hours in a refrigerator at 5 °C. 2.2.3. Microwave synthesis of the composites 95 The rGO/CSO/AgNPs composites preparation procedure was as follows: 17 mg of reduced graphene oxide were mixed with 2 mL of the silver nanoparticles solution (170 µg/mL), 100 µL of the chitosan oligomers solution (22.7 mg/mL) and ethylenglicol (1 mL). Water was then added till a volume of 10 mL was obtained. A microwave treatment was subsequently conducted at 60 ºC or at 120 °C for 10 minutes with stirring (heating ramps of 5 and 10 min, respectively). The obtained 100 products were centrifuged, decanted and washed with ethanol for characterization by analytical techniques. 3 3. Results 105 3.1. Vibrational characterization The vibrational spectra of the rGO/CSO/AgNPs composites prepared by microwave treatment at 60 ºC and 120 ºC are depicted in Figure 1. The two spectra were very similar to each other (only showed small differences in the absorbance intensity), indicating that they corresponded to the same material. Typical peaks such as -NH stretching of CSO, carboxyl C=O and C-O, and alkoxy 110 C-O groups could be readily identified in the spectra, while the peak assigned to epoxy C-O at 1226 cm-1 had disappeared. In addition, amide peaks could be clearly observed at 1636 cm-1 and less clearly at 1412 cm-1. Thus, according to Jiao et al. [10], the FTIR results demonstrate the presence of rGO and the successful synthesis of Ag nanoparticle-containing rGO-based composite materials. 115 4000 3500 3000 2500 2000 1500 1000 500 1500 1400 1300 1200 Absorbance (a.u.) Wavenumber (cm-1) rGO/CSO/AgNPs 60ºC rGO/CSO/AgNPs 120ºC Figure 1. ATR-FTIR spectra of the rGO/CSO/AgNPs composite material 120 3.2. Optical absorption To get an insight into the dynamics of the formation and assembly of rGO/CSO/AgNPs, intermediate UV–vis absorption spectra at different reaction stages were collected (see Figure 2). Whereas the spectrum of rGO revealed an absorption peak centered at 230 nm and a shoulder peak 125 at about 300 nm [17,18], a new peak appeared at about 430 nm, associated to AgNPs decorated onto the rGO/CSO, which implies the formation of rGO/CSO/AgNPs. It is also worth noting that the material obtained at 120 °C showed higher absorbance than that obtained at 60 °C. 130 300 400 500 600 Absorbance (a.u.) Wavelength (nm) rGO/CSO/AgNPs 60 ºC rGO/CSO/AgNPs 120 ºC rGO/AgNPs rGO AgNPs Figure 2. Visible absorption spectra for rGO/CSO/AgNPs composite and its intermediate products 4 3.3. X-ray powder diffraction 135 The XRD pattern of the composite (Figure 3) exhibited a main peak at 2θ ≈ 22º (which, after deconvolution, corresponded to two peaks at d=4.44 Å and d=3.70 Å) followed by a minor peak at 38º (d=2.36 Å). These peaks indicates the presence of the three components of the nano-composite: the peaks at 21º and 24º are to be assigned to CSO and rGO, respectively, in very good agreement with those reported by Matei et al. [19] and Park et al. [20], while the peak at 38º is assigned to 140 AgNPs according to JCPDS patterns [21]. 10 20 30 40 50 60 70 14 16 18 20 22 24 26 28 Intensity (a.u.) 2 (º) rGO/CSO/AgNPs AgNPs (111) d=2.36 A Subtracted Data Cumulative Fit Peak Fit Peak 1 Fit Peak 2 rGO d=3.70 A CSO d=4.44 A Figure 3. XRD pattern of nano-rGO/CSO/AgNPs composite showing CSO, rGO and AgNPs peaks 145 3.4. Imaging of Debye-Scherrer rings On the basis of d-spacing from Debye-Scherrer rings (Figure 4), three assignations could be made: 1.94 Å to (200) reflection of AgNPs; 1.677 to graphite (004) reflections; and 3.237 Å to 150 planar stacking of graphene sheets. Figure 4. Debye-Scherrer rings of nano-rGO/CSO/AgNPs composite 155 3.5. Textural properties and EDS analysis TEM images of a well-dispersed and stabilized sample of the synthesized composite are shown in Figure 5. It could be observed that spherical silver nanoparticles with a diameter ranging from 10 160 to 30 nm were embedded in a matrix of CSO functionalized graphitic rGO sheets and also that no particles were observed outside this matrix. 5 Figure 5. TEM photographs showing the spherical Ag nanoparticles on the CSO functionalized graphitic 165 rGO sheets EDS analysis (Figure 6) further confirmed the claim of the presence of AgNPs in the composite. 170 Figure 6. EDS scan showing the presence of C and Ag elements 175 6 4. Discussion The nano-composite reported in this paper has different characteristics from those of similar composites prepared from GO and rGO with chitosan and silver nanoparticles [8,10]. The main 180 feature of the composite discussed herein is to be a crystalline solid of graphenic texture and not a hydrogel [10]. This difference must be referred to the low proportion of chitosan in our nanocomposite (11.6%) and to the fact that the chitosan is in the form of oligomers, instead of in the form of high or medium molecular weight chitosan. Another important difference is determined by the very low presence of nanosilver in our composite (2%), in contrast to the very high 185 percentages used by other authors, reaching proportions of 88% in weight [8]. Regarding the operating conditions followed in the microwave reactor for synthesizing nanocomposite variants, the results indicate that it is sufficient to apply a program with a temperature as low as 60 °C (for 10 minutes, with stirring) to obtain the desired material. The differences in morphology, properties and yield versus the material obtained at 120 ºC cannot be 190 deemed as significant. 5. Conclusion The facile design and synthesis of rGO/CSO/AgNPs composite material assisted by microwave 195 radiation has been reported. The CS molecule was chosen due to its functional amine segments in the molecular skeleton that can form porous nanostructures via electrostatic interactions and by hydrogen bonding. Morphological characterizations of the obtained composites show the formation of silver nanoaggregates on CSO functionalized rGO sheets by a self-assembly process. The in situ formed silver nanoparticles appeared uniformly anchored on rGO sheets. 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Artículo 3 Control potencial de enfermedades de especies forestales con soluciones de oligómeros de quitosano, propóleo y nanoplata Autores: Iosody Silva-Castro, Jorge Martín-García, Julio Javier Diez, Juan Asdrúbal FloresPacheco, Jesús Martín-Gil y Pablo Martín-Ramos Revista: European Journal of Plant Pathology Editorial: Springer Año: 2017 Volumen: 150 Páginas: 401-411 DOI: 10.1007/s10658-017-1288-4 1 Potential control of forest diseases by solutions of chitosan oligomers, propolis and nanosilver Iosody Silva-Castro1*, Jorge Martín-García2,3, Julio Javier Diez3,4, Juan Asdrúbal FloresPacheco3,4, Jesús Martín-Gil1 and Pablo Martín-Ramos5 5 1 Department of Agricultural and Forestry Engineering, ETSIIAA, University of Valladolid, Avenida de Madrid 44, 34004 Palencia, Spain. 2 Department of Biology, CESAM (Centre for Environmental and Marine Studies), University of Aveiro, Campus Universitario de Santiago, 3810-193 Aveiro, Portugal. 10 3 Sustainable Forest Management Research Institute, University of Valladolid – INIA, Avenida de Madrid 57, 34004 Palencia, Spain. 4 Department of Plant Production and Forestry Resources, ETSIIAA, University of Valladolid, Avenida de Madrid 57, 34004 Palencia, Spain. 5 Department of Agricultural and Environmental Sciences, EPS, Instituto de Investigación en 15 Ciencias Ambientales de Aragón (IUCA), Universidad de Zaragoza, Carretera de Cuarte s/n, 22071 Huesca, Spain. * Corresponding author at: Agriculture and Forestry Engineering Department, ETSIIAA, University of Valladolid, Avenida de Madrid 44, 34004 Palencia, Spain. Email: [email protected] 20 Abstract There is a growing necessity to replace chemical agents with ecofriendly materials, arising from their impact on the environment and/or human health, which calls for the design of new broad25 spectrum fungicides. In this work, chitosan oligomers (COs), propolis (Ps) and silver nanoparticles (AgNPs) mixtures in solution were assessed to control the growth of different phytopathogenic fungi and oomycetes in vitro. Binary solutions of COs-Ps and COs-AgNPs evinced the highest antifungal effect against Fusarium circinatum and Diplodia pinea fungi, respectively, with a ca. 80% reduction in their mycelial growth. The COs solution by itself also proved to be greatly effective against 30 Gremmeniella abietina, Cryphonectria parasitica and Heterobasidion annosum fungi, causing a reduction of 78%, 86% and 93% in their growth rate, respectively. Likewise, COs also attained a 100% growth inhibition on the oomycete Phytophthora cambivora. On the other hand, Ps inhibited totally the growth of Phytophthora ×alni and Phytophthora plurivora. The application of AgNPs reduced the mycelial growth of F. circinatum and D. pinea. However, the AgNPs in some binary and 35 ternary mixtures had a counter-productive effect on the anti-fungal/oomycete activity. In spite of the fact that the anti-fungal/oomycete activity of the different treatments showed a dependence on the particular type of microorganism, these solutions based on natural compounds can be deemed as a promising tool for control of tree diseases. 2 40 Keywords: anti-fungal; anti-oomycetes; forest pathogens; natural compounds. 1. Introduction Phytopathogenic microorganisms are responsible for major economic losses and ecological impacts, affecting from seedling nurseries to mature trees in plantations, seed orchards, landscape 45 plantings, or native forests (Hirooka and Ishii 2013; Gordon et al. 2015). All over the world, several species of conifers are affected by common ascomycete fungi, such as Fusarium circinatum Nirenberg & O'Donnell, responsible for pitch canker disease (Wingfield et al. 2008); Diplodia pinea (Desmaz.) J. Kickx fil. (= Sphaeropsis sapinea (Fr.) Dyko & Sutton), which causes Diplodia tip blight and stem canker disease (Gibson 1979; Adamson et al. 2015); and Gremmeniella abietina 50 (Lagerberg) Morelet (anamorph: Brunchorstia pinea (P. Karsten) Höhnel) that produces shoots dieback and cankers on stems and trunks (Kaitera and Jalkanen 1992; Romeralo et al. 2015), causing the death of conifers including spruce, fir, larch, pine and juniper. In the same way, another of the most important pathogens in coniferous forests is Heterobasidion annosum (Fr.) Bref. (= Fomes annosus (Fr.) Cooke) basidiomycete, which causes root and butt rot (Asiegbu et al. 2005; Garbelotto 55 and Gonthier 2013). Other main forest pathogens include Cryphonectria parasitica, one of the most undesirable introduced plant pathogens, which causes chestnut blight on species in the genus Castanea (Heiniger and Rigling 1994; González-Varela et al. 2011); and oomycetes species such as Phytophthora. These latter comprise P. cambivora (Petri) Buisman, also a common pathogen of Castanea, Fagus and other 60 hardwoods (Jung et al. 2005); P. ×alni (Brasier & S.A. Kirk) Husson, Ioos & Marçais, nothosp. nov., which cause alder decline by dieback, small sparse and yellowish leaves, excessive fructification, and tarry and rusty exudates (Husson et al. 2015); and P. plurivora T. Jung and T.I. Burgess, which causes aerial canker and collar rot in several species, including beech, oaks and alders (Jung and Burgess 2009; Haque et al. 2014; Haque et al. 2015). 65 To date, control of plant diseases has typically been performed by application of high toxic chemicals, whose excessive use has occasioned undesired impacts on the environment and on human health (Hirooka and Ishii 2013). Moreover, regulations are increasingly limiting the utilization of high toxic chemicals and promoting the use of integrated pest management and non-chemical alternatives to pesticides (Directive 2009/128/EC). 70 Chitosan is a natural polymer composed of randomly distributed β-(1-4) D-glucosamine and Nacetyl-D-glucosamine units. It can be found in the form of chitin in the shells of crustaceans and in 9 240 Figure 3. Radial growth rate and interaction among treatments based on chitosan oligomers (COs), Propolis and silver nanoparticles (AgNPs) against (a) F. circinatum; (b) D. pinea; (c) G. abietina; (d) C. parasitica; and (e) H. annosum fungi. Different letters above bars indicate significantly different means (generalized Welch procedure 0.2 trimmed means, a = 0.05). Error bars show the standard deviation. Note: Only significant interactions from the Post hoc analyses are shown. 245 3.3. Anti-oomycete activity As regards the assays conducted with oomycetes (Figure 4), a remarkable inhibitory activity was attained for COs and Ps. Treatments against P. cambivora (Figure 4a) evidenced an interaction among 250 the three agents (F=64.1, p=0.001), but all treatments with COs (individual, binary and ternary mixtures) presented 100% of growth inhibition. The treatment with the individual Ps solution also showed growth inhibition (43%), but AgNPs and Ps-AgNPs treatments did not exhibit any significant differences vs. the control. On the other hand, an interaction between COs and Ps was found in treatments against P. ×alni 255 and P. plurivora (F=23, p=0.002 and F=722.8, p=0.001, respectively). While the application of COs and Ps (individual or mixed) resulted in a similar growth inhibition for P. ×alni, the addition of Ps played a leading role in the growth inhibition for P. plurivora (Figure 4b and Figure 4c). 260 10 Figure 4. Radial growth rate and interaction among treatments based on chitosan oligomers (COs), Propolis and silver nanoparticles (AgNPs) against (a) P. cambivora; (b) P. ×alni; and (c) P. plurivora oomycetes. Different letters above bars indicate significantly different means (generalized Welch procedure 0.2 trimmed 265 means, a = 0.05). Error bars show the standard deviation. Note: Only significant interactions from the Post hoc analyses are shown. 4. Discussion 270 The present study has demonstrated that the three compounds (COs, Ps and AgNPs) have an antifungal effect on different forest pathogens. COs by itself showed an inhibitory effect on the mycelial growth of all pathogens tested. Although the antifungal activity of chitosan polymer has been already reported by other authors both in in vitro and in vivo experiments, for example, chitosan 275 applications to increase the resistance of pine seedlings to F. circinatum and D. pinea (Reglinski et al. 2004; Fitza et al. 2013), this study confirms the importance of the use of low molecular weight chitosan such as COs. It is worth noting that when chitosan with higher molecular weight than that used in this study (e.g., 50,000 Da instead 2,000 Da) are applied, a lower antifungal activity is attained, with 35% of reduction of mycelial growth of D. pinea in the first day as reported by Singh 280 11 et al. (2008). This is consistent with the results reported by Avelelas et al. (2014), Qiu et al. (2014) and Cobos et al. (2015), who demonstrated that chitosan antifungal activity increased in inverse proportion to its molecular weight. Consequently, COs of molecular weight under 2,000 Da, might be a preferable option as compared to commercial ‘low molecular weight’ chitosan (i.e., 50,000 to 190,000 Da, CAS Number 9012-76-4) in terms of its activity against D. pinea. On the other hand, in 285 an in vitro study by Ziani et al. (2009), the use of chitosan solutions proved to be more effective against Aspergillus niger, Alternaria alternata and Rhizopus oryzae than the use of films, where presumably, the chitosan solution had positive charges on the quaternary amino groups that interacted with the fungal cell walls, while for the films a protonation loss occurred. The inhibitory effect of Ps was demonstrated on most of the pathogens tested (F. circinatum, D. 290 pinea, C. pararisitica, P. cambivora, P. ×alni and P. plurivora). The use of propolis has not been as well studied as chitosan, although its inhibition capacity against F. circinatum was already reported by Iturritxa et al. (2013). However, they reported a fungicidal effect, whereas in this study a growth inhibition effect was found. The application of AgNPs by itself also reduced the mycelial growth of F. circinatum and D. 295 pinea, which is consistent with the results reported by Narayanan and Park (2014), who observed slight to moderate inhibition against wood-degrading fungi when a low dose of AgNPs was used. Nevertheless, AgNPs had not a significant anti-oomycete activity on the species tested in this study, contrasting with Mahdizadeh et al. (2015), who found that another oomycete (Pythium aphanidermatum (Edson) Fitzp.) was the most sensitive pathogen to nanosilver among the six tested 300 species. The effect of the binary solutions of the compound tested varies according to the species. While the COs-Ps binary solution showed the highest antifungal effect against F. circinatum, the result of the application of AgNPs in the binary solutions varies according to the pathogen. Indeed, the binary solution COs-AgNPs recommended by Wang et al. (2015) was the most promising mixture in order 305 to control D. pinea. Nevertheless, the use of COs-AgNPs and Ps-AgNPs solutions had a counterproductive effect on the anti-fungal/oomycete activity against G. abietina and P. cambivora, respectively. This is in contrast to other studies in which nanosilver was also incorporated into chitosan, although in higher doses. For example against ascomycete Colletotrichum gloeosporioides (Penz.) Penz. & Sacc., the mixture showed excellent results: the inhibitory action increased from 44% 310 to 100% as the AgNPs concentration was increased from 0.1 up to 100.0 μg/mL (Chowdappa et al. 2014). It is also noteworthy that the solution consisting only of AgNPs did not show statistically significant difference vs. the control treatment, in contrast to the study by Narayanan and Park (2014), 12 who observed slight to moderate inhibition against wood-degrading fungi when a low dose of AgNPs was used. In the same vein, Saharan et al. (2013) and Saharan et al. (2015) found that the nanocopper315 chitosan complex showed growth inhibition against other ascomycota such as Fusarium oxysporum and A. alternata. They suggested that addition of nanometals increased the surface charge density and provided more electrostatic interaction with fungal membrane. Differences in the inhibitory behavior of the similar COs-Ps-AgNPs mixture have been reported for other fungal species and different application procedures: the COs-Ps-AgNPs ternary complex did 320 not improve the antifungal/anti-oomycete activity compared to the binary solutions in this study, which contrasts with the complete inhibition obtained using similar COs-Ps-AgNPs mixtures applied to D. seriata and Bipolaris oryzae (Breda de Haan) Shoemaker (Matei et al. 2015; Araujo-Rufino et al. 2016). This discrepancy may be associated to that the gel phase used was ascribed to the higher concentrations of chitosan oligomers in the gel (20-25 mg/mL) vs. the aqueous solution of this study 325 (1 mg/mL). The bands in the ATR-FTIR spectrum of the COs-Ps-AgNPs composite evidenced a weak interaction among COs and AgNPs, even weaker than that reported for chitosan-AgNPs thin films and nanocomposites manufactured by spin-coating (Wei et al. 2009; Wang et al. 2015), whose infrared spectra showed shifts between 5 and 10 cm-1. On the other hand, the spectrums of COs and 330 COs-Ps showed very similar bands to those reported in other works for chitosan (Matei et al. 2015; Stroescu et al. 2015; Branca et al. 2016) and propolis extracts (Franca et al. (2014); Siripatrawan and Vitchayakitti (2016). A differential feature of this investigation in comparison to the literature was that in the preparations described above Green Chemistry procedures were used, without need for the addition 335 of chemical bond reinforcing agents, widely used in other works (Gu et al. 2014; Jemec et al. 2016). Accordingly, these eco-friendly compounds could be useful in management strategies based on integrated approach, for example in the use of appropriate nursery hygiene practices. Likewise, the application of chitosan had been suggested using the chitosan-based Biochikol 020 PC, a biological agent with fungicidal properties and resistance stimulator, in order to control P. xalni complex in 340 forest nurseries (Oskazo 2007). In conclusion, from the results of the in vitro growth inhibition experiments respect the antifungal/oomycete effect of individual, binary and ternary mixtures of COs, Ps and AgNPs, assayed against eight plant pathogens, it could be inferred that: (i) the inhibitory activity against fungi and oomycetes of the individual low molecular weight COs solutions was significantly high (reaching 345 growth rate reductions of up to 78, 86, 93% and 100% against G. abietina, C. parasitica, H. annosum 13 and P. cambivora, respectively); (ii) the growth inhibition is enhanced by association of COs with Ps (e.g., F. circinatum) and COs with AgNPs (e.g. D. pinea); and (iii) the COs-P-AgNPs ternary complex did not improve the antifungal/anti-oomycete activity compared to the binary solutions. Thus, the weak interactions that appear in solution amongst the three components (evidenced by FTIR) 350 suggested that strong interactions are necessary to achieve the desired anti-fungal/oomycete effect. Additionally, further studies are essential to determine the effect of the COs-Ps-AgNPs combinations on seeds, tree seedlings and mature trees infested by different pathogens, as an innovative application system useful in an integrated management approach. 355 5. Acknowledgments This article is based upon work from COST Action FP1406 PINESTRENGTH (Pine pitch canker - strategies for management of Gibberella circinata in greenhouses and forests), supported by COST (European Cooperation in Science and Technology) and project AGL2015-69370-R 360 (MINECO/FEDER) funded by the Spanish Ministerio de Economía y Competitividad and the Fondo Europeo de Desarrollo Regional (FEDER). Calabazanos Forest Health Center - Junta de Castilla y León (Villamuriel de Cerrato, Palencia, Spain) is gratefully acknowledged for supplying the Cryphonectria parasitica, Heterobasidion annosum fungi and the Phytophthora cambivora oomycete. I. 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Web references 420 www.ico.org/trade_statistics.asp Artículo 5 Aplicación de recubrimientos bioactivos de quitosano y propóleo para la protección de especies de pinos contra Fusarium circinatum Autores: Iosody Silva-Castro, Julio Javier Diez, Pablo Martín-Ramos, Glória Pinto, Artur Alves, Jesús Martín-Gil y Jorge Martín-García Revista: Forests Editorial: MDPI Año: 2018 Volumen: 9 Issue: 11 Páginas: 685-699 DOI: https://doi.org/10.3390/f9110685 Forests 2018, 9, x; doi: FOR PEER REVIEW www.mdpi.com/journal/forests Article Application of Bioactive Coatings Based on Chitosan and Propolis for Pinus spp. Protection against Fusarium circinatum Iosody Silva-Castro 1,*, Julio Javier Diez 2,3, Pablo Martín-Ramos 4, Glória Pinto 5, Artur Alves 5, 5 Jesús Martín-Gil 1 and Jorge Martín-García 2,5 1 Department of Agricultural and Forestry Engineering, ETSIIAA, University of Valladolid, Avenida de Madrid 44, 34004 Palencia, Spain; [email protected] 2 Sustainable Forest Management Research Institute, University of Valladolid—INIA, Avenida de Madrid 57, 34004 Palencia, Spain; [email protected] (J.J.D.); [email protected] (J.M.-G.) 10 3 Department of Plant Production and Forestry Resources, ETSIIAA, University of Valladolid, Avenida de Madrid 44, 34004 Palencia, Spain 4 Department of Agricultural and Environmental Sciences, EPS, Instituto Universitario de Investigación en Ciencias Ambientales de Aragón (IUCA), Universidad de Zaragoza, Carretera de Cuarte s/n, 22071 Huesca, Spain; [email protected] 15 5 Department of Biology & CESAM (Centre for Environmental and Marine Studies), University of Aveiro, Campus Universitario de Santiago, 3810-193 Aveiro, Portugal; [email protected] (G.P.); [email protected] (A.A.) * Correspondence: [email protected] Received: 25 September 2018; Accepted: 26 October 2018; Published: date 20 Abstract: Pine pitch canker (PPC) is a major threat to pine forests worldwide because of the extensive tree deaths, reduced growth, and degradation of timber quality caused by it. Furthermore, the aggressive fungus responsible for this disease (Fusarium circinatum) can also infect pine seeds, causing damping-off in young seedlings. This study proposes an approach based on coating treatments consisting of natural products to ensure seed protection. Seeds from two pine 25 species (the most sensitive to this disease, Pinus radiata D. Don, and a more resistant one, Pinus sylvestris L.) were coated with single and binary mixtures of low and medium molecular weight chitosan and/or ethanolic-propolis extract. The germination rate, preand post-emergence mortality, total phenolic content, and radical scavenging activity were assessed. All treatments, and especially the one based on chitosan oligomers, had a beneficial impact on P. sylvestris seedlings, 30 significantly enhancing survival rates and displaying a positive influence on the total phenolic content and on the seedlings’ radical scavenging activity. Conversely, non-significant negative effects on germination percentages were observed in the case of P. radiata seeds. The proposed treatments show promise for the protection of P. sylvestris seedlings against PPC. Keywords: antifungal; antioxidant; natural coating; seed protection; total phenolic content 35 1. Introduction Fusarium circinatum Nirenberg & O’Donnell is a quarantine fungus according to the European and Mediterranean Plant Protection Organization (EPPO) [1] that causes pine pitch canker (PPC) and which has been deemed as one of the most damaging pathogens for Pinus spp. throughout the 40 world [2]. In forest nurseries, F. circinatum causes preand post-emergence damping-off, wilting of seedlings, shoot and tip dieback, and it finally leads to the death of the infected seedlings [3]. F. circinatum can be found in nurseries of North and South America, South Africa, Asia, and Southern Europe [4]. The use of seeds from orchards poses a serious threat of spread of this fungus to Forests 2018, 9, x FOR PEER REVIEW 2 of 14 nurseries worldwide [5]. At present, there are no effective means of controlling PPC in nurseries and 45 forest plantations. An integrated management plan should therefore include both adequate quarantine measures and appropriate nursery and silvicultural management strategies. Thus, the implementation of seed protection in nursery health practices would be of paramount importance. Seed-coating technology may act not only as a phytosanitary against pests and diseases but may also enhance germination rates and crop yield [6]. Chemicals such as imidacloprid and 50 tebuconazole [7,8] have been extensively used as coatings for seeds, but nowadays their use in forests is highly restricted (Directive 2009/128/EC). Consequently, natural substances and biological agents—such as starch [9] and Trichoderma spp. [10,11], respectively—are receiving increasing attention as environmentally friendly alternatives [12,13]. Amongst these natural products, chitosan and propolis have shown great promise for plant 55 protection purposes, and, in particular, against F. circinatum [14–16]. Chitosan, obtained from chitin’s deacetylation, is an organic polymer with a cationic character, which confers numerous physicochemical and biological properties, such as copolymerization, filmogenicity, biocompatibility, biodegradability, and also antibiotic properties [17–20]. In turn, propolis is a chemically very complex resinous bee product with many biological properties [21]. Due to its 60 composition, mainly flavonoids and phenolic acids, it is able to alter membrane permeability and inhibit protein synthesis in microorganisms [22]. For agricultural applications, chitosan is applied as an elicitor (inducer of plant resistance) and antifungal product because of its ability to induce the synthesis of phenolic compounds [23], which are involved in tolerance mechanisms against biotic or abiotic stressors [24]. Among the large 65 number of phenolic antioxidants, flavonoids, for instance, can directly inhibit microbial enzymes production [25]. Other specific flavonoids, such as anthocyanins, are known to increase the antioxidant activity, reducing the susceptibility to fungi [26]. The total phenolic content (TPC) and/or the radical scavenging activity (RSA) are properties commonly analyzed in order to identify responses caused by elicitors in plants [27,28]. 70 The molecular weight of chitosan plays a key role in its fungicide properties [29], in such a way that low molecular weight chitosan (i.e., oligomers) is more effective at inducing a set of plant defense responses than its higher molecular weight counterpart (i.e., polymers) [30]. In spite of the fact that chitosan oligomers feature better antimicrobial activity than high molecular weight chitosan [31,32], the later has a higher viscosity [33], which explains why it is more frequently used as a 75 coating [34,35]. In view of the chemical affinity and well-established synergies between chitosan and propolis [36–38], the main aim of the work reported here was to evaluate the protection conferred by bioactive seed coatings based on chitosan with two different molecular weights—medium (CMMW) and low (CLMW)—and propolis ethanolic extract (PEE) composites against F. circinatum in the 80 relatively resistant Pinus sylvestris L. and in the highly susceptible Pinus radiata D. Don. 2. Materials and Methods 2.1. Fungal and Plant Materials The Fusarium circinatum isolate FcCa6 used in this study was obtained from the collection of the Forest Entomology and Pathology Laboratory at the University of Valladolid, Spain [39–43]. Plant 85 material consisted of seeds of P. radiata and P. sylvestris (see provenance in Table 1). Table 1. Provenance of plan material. Seed Species Provenance Provided by Pinus radiata (Monterey pine) “Galicia montañas meseta Interior” (Spain) Consellería do Medio Rural (Xunta de Galicia, Spain) Pinus sylvestris (Scots pine) “Sierra de Guadarrama” (Spain) El Serranillo Nursery (Ministry of Agriculture and Environment, Spain) Forests 2018, 9, x FOR PEER REVIEW 3 of 14 2.2. Seed-Coating Preparation 2.2.1. Reagents In order to obtain the coating material, medium molecular weight chitosan powder, purchased 90 from Hangzhou Simit Chemical Technology Co. (Hangzhou, China), and propolis with a content of poly-phenols and flavonoids of ca. 10% (w/v) from Burgos (Spain) were used. High specific surface (70–85 m2/g) halloysite in powder form, from Dunino mine, with reduced iron content (ca. 5%), was purchased from Intermark (Gliwice, Poland). All other reagents (viz., acetic acid, hydrochloric acid, hydrogen peroxide, ethanol, Tween 80, Folin–Ciocalteu reagent, 2,2-diphenyl-picrylhydrazyl, etc.) 95 were of analytical grade and were purchased from Sigma-Aldrich Química S.L. (Madrid, Spain). 2.2.2. Preparation of the Seed Coating Solutions Due to differences in viscosity, and therefore in the adhesion to the seed surface, chitosan with two different molecular weights was assessed. The medium molecular weight chitosan (CMMW 60–130 kDa) was prepared by dissolving 2 g of commercial chitosan in 100 mL of acetic acid solution 100 (1% v/v) under constant stirring at 60 °C for 2 h until its complete dissolution. To obtain the low molecular weight chitosan (CLMW 20 kDa), it was necessary to add hydrogen peroxide (0.3 M) to the chitosan solution obtained in the previous step, keeping the same conditions until a brown and less viscose solution was obtained after 1 h [44]. Propolis ethanolic extract (PEE) composites were prepared by introducing the finely grinded resin into a hydroalcoholic solution (7:3 v/v). After 105 stirring for 72 h at room temperature, the insoluble particles were filtered [45]. To obtain the first composite (CMMW-PEE), Tween 80 was added dropwise to a 10 mg·mL−1 chitosan solution, followed by the addition of 1 mg·mL−1 of propolis solution. The mixture was sonicated with a probe-type UIP1000hdT ultrasonicator (Hielscher, Teltow, Germany; 1000 W, 20 kHz) for 3 min in cycles of 1 min with sonication and 1 min without sonication to keep the 110 temperature below 40 °C [37]. To obtain the second composite (CLMW-PEE), a similar process was followed, albeit replacing Tween 80 with halloysite, a natural clay innocuous to seeds and fungus. Halloysite was added to the less viscous solutions (CLMW, PEE and CLMW-PEE) in order to improve their adherence to the surface of the seeds. 2.2.3. Seed Coating Application 115 Prior to coating application, seeds underwent the following pre-germination procedure according to Martín-García et al. [40]: they were initially soaked in water for 24 h (renewing the water after 12 h), followed by soaking in hydrogen peroxide (3%) for 15 min, triple-washing with sterile distilled water, and an immersion in sterile distilled water for another 30 min (in order to clear away any remaining hydrogen peroxide). Subsequently, the seeds were placed in a laminar flow 120 hood in order to dry them. Six treatments were applied: (1) Control (sterile water), (2) CMMW, (3) CLMW, (4) PEE, (5) CMMW-PEE, and (6) CLMW-PEE. Halloysite (1 g to 100 mL of solution) was applied in treatments 3, 4, and 6. Then, the seeds were dried again to form a film on their surface (Figure 1) and were kept in sterile flasks until sowing. Seventy seeds (replicates) of each pine species were prepared per 125 treatment (i.e., a total of 840 seeds). Forests 2018, 9, x FOR PEER REVIEW 4 of 14 Figure 1. Film formation on seed surface of P. radiata seeds: (a) control (sterile water), (b) coating based on medium molecular weight chitosan (CMMW) and (c) coating based on low molecular weight chitosan (CLMW) with halloysite. 130 2.3. Pathogenicity Tests Following the procedure presented in Martín-García et al. [41], a spore suspension of F. circinatum (Fc) was cultured on potato dextrose broth (PDB). Five mycelial agar plugs (5 mm in diameter) were added to 1 L of PDB and were placed on an orbital shaker at 180 cycles for 24 h at 25 °C. After that, the liquid medium was filtered twice through sterile cheesecloth to remove hyphae 135 and the spore concentration was adjusted to 1 × 103 spores·mL−1 with a Neubauer hemocytometer. Seventy seeds per each type of coating (plus 70 without coating) and per pine species were individually sown in germination trays (96 mL) containing a twice-autoclaved (105 kPa, 120 °C, 30 min) mixture of peat and vermiculite (1:1, v/v). For half of the seeds with each type of coating, the spore suspension of F. circinatum was added to the substrate when the seeds were sown. The other 140 half of the seeds were mock-inoculated with sterile distilled water. Thus, the experimental design consisted of twelve treatments: (i) control, (ii) CMMW, (iii) CLMW, (iv) PEE, (v) CMMW-PEE, (vi) CLMW-PEE, (vii) Fc, (viii) CMMW-Fc, (ix) CLMW-Fc, (x) PEE-Fc, (xi) CMMW-PEE-Fc, and (xii) CLMW-PEE-Fc. Incubation of the germination trays was conducted in a growth chamber under controlled 145 conditions (temperature: 21.5 °C; photoperiod: 16/8 h light/dark). They were watered every two days, with equal water doses, all over the period of study. Seed germination and subsequent seedling mortality were monitored on a daily basis. 2.4. Determination of Total Phenolic Content (TPC) and Radical Scavenging Activity (RSA) The extracts were obtained according to the following process: four asymptomatic seedlings 150 were collected from each of the trays 30 days after the sowing date, including control (i) and inoculated treatments (vii) to (xii). These seedlings were dried (40 °C, 7 days) and grinded. Then, 20 µg of each sample in powder form was added to 2 mL of methanol (70%, vol.) acidified with a few drops of HCl (1 M). The mixture (methanol and sample) was kept in shaking condition for 2 h and filtered to get the extracts [27] that were used in both analyses (TPC and RSA). 155 The TPC was evaluated with a modified Folin–Ciocalteu procedure [46]: firstly, 100 µL of the sample methanolic extract was mixed with 450 µL of distilled water and 50 µL of Folin–Ciocalteu reagent. After 10 min, 400 µL of Na2CO3 was added and the samples were kept in dark for 90 min. The absorbance was measured at 765 nm using a Thermo Scientific Multiscan Go Microplate Spectrophotometer (Waltham, MA, USA). A calibration curve was prepared with standard gallic 160 acid (y = 0.0407x − 0.0595; r2 = 0.99) and used to express the results as gallic acid equivalents (GAE, in mg of gallic acid per mL of extract). Forests 2018, 9, x FOR PEER REVIEW 5 of 14 The RSA was determined by the 2,2-diphenyl-picrylhydrazyl (DPPH) method, according to the procedure described by Chiang et al. [47], with some modifications. Briefly, 50 µL DPPH radical solution (1 × 10−4 M) was added to 450 µL of the sample methanolic extract, and the reaction mixtures 165 were kept at room temperature for 30 min. Absorbance was recorded at 517 nm. The radical scavenging activity was expressed as a percentage (RSA%) relative to the control, using the following equation: RSA% = [(Ablank − Asample)/Ablank] × 100 (1) where Ablank is the absorbance of the blank (distilled water) and Asample is the absorbance of the sample extracts. 170 2.5. Statistical Analyses All analyses were performed using R software environment (R Foundation for Statistical Computing, Vienna, Austria). Chi-square tests (χ2) were carried out using the mock-inoculated treatments—(i) to (vi)—to test the effect of the coatings on germinative capacity. Likewise, chi-square tests (χ2) were carried out using the control (i) and inoculated treatments—(vii) to 175 (xii)—to test the protective effect of seed coatings on the pre-emergence mortality caused by F. circinatum. To prevent overestimation of statistical significance for small data, Yates’ correction for continuity was applied for counts smaller than 5. To test the post-emergence mortality up to the end of the experiment (when no seedling from Fc treatment (vii) was alive, 40 and 30 days after sowing for P. sylvestris and P. radiata, respectively; in the case of P. radiata, the last four living seedlings were 180 removed to carry out the TPC and RSA analyses), a survival analysis based on the Kaplan–Meier non-parametric estimator [48] was carried out using “Survival” package [49]. “Sirvfit” and “Survdiff” functions, also available in the same package, were used to create survival curves and to analyze the differences between the curves, respectively. Analyses of variance (ANOVAs) and Tukey’s HSD (honestly significant difference) post-hoc test were carried out to assess the effect on 185 TPC and RSA of seedling from control (i) and inoculated with F. circinatum treatments—(vii) to (xii)—as a function of the seed coatings at 30 days after sowing. These analyses were only performed in the pine species (P. sylvestris) in which changes on mortality rates as a result of seed coating were demonstrated in the previous step. All analyses were performed using R software environment (R Foundation for Statistical Computing, Vienna, Austria). 190 3. Results 3.1. Germination Test Data on the germination percentage (GP) for each of the species and coatings is shown in Figure 2. The highest GP (85.7%) for P. sylvestris was attained for the control and PEE treatments. The use of chitosan, either individually or in combination with PEE, did not enhance the GP. In fact, CMMW and 195 CMMW-PEE treatments led to lower GP values (60% and 62.9%, respectively) than that of the control treatment (Figure 2a). In the case of P. radiata seeds, the use of chitosan and propolis did not enhance the GP either, and the binary CLMW-PEE composite decreased the GP vs. the control treatment (Figure 2b). No significant differences in terms of GP were found between Monterey pine and Scots pine (χ2 = 1.43, p = 0.23). 200 Forests 2018, 9, x FOR PEER REVIEW 6 of 14 Figure 2. Germination percentage (GP) of (a) P. sylvestris and (b) P. radiata seeds coated with medium and low molecular weight chitosan (CMMW and CLMW, respectively) and/or with propolis ethanolic extract (PEE) at 40 and 30 days after sowing, respectively. Averages with the same letter were not significantly different according to the Chi-square test (χ2) (α ≤ 0.05). 3.2. Pathogenicity Test 205 Inoculations with F. circinatum did not cause pre-emergence mortality for either P. sylvestris (χ2 = 0.85, p = 0.36) or for P. radiata (χ2 = 0.85, p = 0.36). Nonetheless, survival analyses demonstrated significant differences in post-emergence mortality among treatments in P. sylvestris (χ2 = 49.7, p < 0.001). No seedlings of the Fc treatment survived beyond 40 days after inoculation (dai), whereas no mortality was recorded in the control seedlings. All coating treatments improved the survival of P. 210 sylvestris inoculated seedlings, resulting in survival rates of over 50% for the CMMW-Fc, CLMW-Fc, and CMMW-PEE-Fc treatments. Chitosan-only treatments (CLMW-Fc and CMMW-Fc) showed similar protection efficacies as PEE-Fc (χ2 = 1.4, p = 0.23; and χ2 = 1.3, p = 0.26, respectively). However, in the CMMW-PEE-Fc and CLMW-PEE-Fc binary composites, the addition of PEE did not improve the survival rate in comparison to CMMW-Fc and CLMW-Fc (χ2 < 0.001, p = 0.88; and χ2 = 3.2, p = 0.07, 215 respectively) (Figure 3a). Mortality of inoculated P. radiata seedlings was faster than that of P. sylvestris seedlings. In fact, just four seedlings from the non-coated seeds inoculated with F. circinatum survived until 30 dai. Significant differences in post-emergence mortality were found between control treatment and inoculated seedlings, regardless of the coating treatment (χ2 = 67.7, p < 0.001). However, none of the 220 coating treatments was able to significantly improve the survival of the inoculated seedlings, neither the individual components CMMW-Fc, CLMW-Fc, and PEE-Fc (χ2 = 1.7, p = 0.19; χ2 = 0.2, p = 0.64; and χ2 Forests 2018, 9, x FOR PEER REVIEW 7 of 14 = 0.2, p = 0.66, respectively) nor the CMMW-PEE-Fc and CLMW-PEE-Fc binary composites (χ2 = 2.5, p = 0.11; and χ2 = 0.2, p = 0.69, respectively) (Figure 3b). Figure 3. Plot of survival probability, determined using the Kaplan-Meier estimate of the survival 225 function, for (a) P. sylvestris and (b) P. radiata seedlings inoculated with Fusarium circinatum (Fc) as a function of the seed coating treatments. CMMW and CLMW stand for medium and low molecular weight chitosan, respectively, PEE stands for propolis ethanolic extract. No mortality was registered for mock-inoculated and coating treatments (i.e., (ii) to (vi)). These curves were not shown to avoid multi-overlaps with the curve of the control treatment. Averages with the same letter were not 230 significantly different according to the Kaplan–Meier estimator (α ≤ 0.05). 3.3. Total Phenolic Content and Radical Scavenging Activity The TPC varied significantly in P. sylvestris seedlings as a function of the coating treatment (F = 4.99, p < 0.01). The mean TPC of the control treatment was 6.8 mg·mL−1 of GAE, which was significantly higher than the value obtained in the Fc treatment (2.7 mg·mL−1), evidencing a 235 significant TPC reduction caused by the pathogen (Figure 4a). TPC values did not change in inoculated-coated seeds in comparison to the control treatment, with the exception of the CMMW-Fc treatment. In particular, the CLMW-Fc treatment led to a TPC value comparable to that of the control treatment. Forests 2018, 9, x FOR PEER REVIEW 8 of 14 The negative effect caused by the pathogen was also evidenced in the low antioxidant capacity 240 of inoculated seedlings in comparison to the high antioxidant activity shown by the control treatment (83.5%). This RSA value was just preserved by the CLMW-Fc and PEE-Fc treatments, which did not vary significantly in comparison with the control treatment (Figure 4b). However, CMMW and the binary composites did not succeed in reverting the aforementioned negative effect exerted by the pathogen. 245 Figure 4. (a) Total phenolic contents (TPC) in gallic acid equivalents (GAE, mg·mL−1) and (b) percentage of radical scavenging activity (RSA%) of P. sylvestris seedlings from control (i) and inoculated with Fusarium circinatum (Fc) treatments—(vii) to (xii)—as a function of the seed coatings at 30 days after sowing. CMMW and CLMW stand for medium and low molecular weight chitosan, respectively, PEE stands for propolis ethanolic extract. Averages with the same letter were not 250 significantly different according to the Tukey’s HSD test (α ≤ 0.05). 4. Discussion At least 60 species of Pinus along with Pseudotsuga menziesii (Mirb.) Franco are known to be susceptible to PPC. Amongst them, P. radiata is recognized as the most susceptible [3], while P. sylvestris has also been reported to present a high susceptibility [41,43,50–53]. In this study, seed 255 coatings based on chitosan—with two different molecular weights—and propolis were applied in order to test if these bioactive products were able to confer resistance against F. circinatum in the two species mentioned above. Artículo 6 Recubrimientos a base de quitosano para prevenir la descomposición de madera de Populus spp. causada por Trametes Versicolor Autores: Iosody Silva-Castro, Milagros Casado-Sanz, Agustín L. Alonso-Cortés, Pablo Martín-Ramos, Jesús Martín-Gil y Luis Acuña-Rello Revista: Coatings Editorial: MDPI Año: 2018 Volumen: 8 Páginas: 415-430 DOI: https://doi.org/10.3390/coatings8120415 Coatings 2018, 8, x; doi: FOR PEER REVIEW www.mdpi.com/journal/coatings Article Chitosan-based coatings to prevent the decay of Populus spp. wood caused by Trametes versicolor Iosody Silva-Castro 1, Milagros Casados-Sanz 2, Agustín L. Alonso-Cortés 3, Pablo Martín-Ramos 4, Jesús Martín-Gil 1 and Luis Acuña-Rello 2,* 5 1 Environmental Technology Laboratory, Department of Agricultural and Forestry Engineering, University of Valladolid, Avda. de Madrid 44, 34004 Palencia, Spain; io[email protected] (I.S.-C.); [email protected] (J.M-G.) 2 Wood Technology Laboratory, Department of Agricultural and Forestry Engineering, University of Valladolid, Avda. de Madrid 44, 34004 Palencia, Spain; [email protected] (M.C.-S.); [email protected] (L.A.- 10 R.) 3 Biotechnology Laboratory, Department of Agricultural and Forestry Engineering, University of Valladolid, Avda. de Madrid 44, 34004 Palencia, Spain; [email protected] (A.L.A.-C.) 4 Department of Agricultural and Environmental Sciences, EPS, Instituto Universitario de Investigación en Ciencias Ambientales de Aragón (IUCA), Universidad de Zaragoza, Carretera de Cuarte s/n, 22071 15 Huesca, Spain; [email protected] (P.M.-R.) * Corresponding author: [email protected]; Tel: +34 (979) 108397; Fax: +34 (979) 108397 Abstract: Chitosan and chitosan oligomers are receiving increasing attention due to their antimicrobial properties. In the present study, they were assayed as a preventive treatment against white-rot decay of Populus wood (very important in economic and environmental terms), caused by 20 Trametes versicolor fungus. Their capacity to incorporate different chemical species into the polymer structure with a view to improving their anti-fungal activity was also assessed by mixing oligochitosan with propolis and silver nanoparticles. The minimum inhibitory concentration of mediummolecular weight chitosan (MMWC), chitosan oligomers (CO), propolis (P), nanosilver (nAg), and their binary and ternary composites against T. versicolor was determined in vitro. Although all 25 products exhibited anti-fungal properties, composites showed an enhanced effect as compared to the individual products: 100% mycelial growth inhibition was attained for concentrations of 2.0 mg·mL-1 and 0.2 mg·mL-1 for the CO-P binary mixture, respectively; and 2 µg·mL-1 for nAg in the ternary mixture. Subsequently, MMWC, CO, CO-P and CO-P-nAg composites were tested on poplar wood blocks as surface protectors. Wood decay caused by the fungus was monitored by 30 microscopy and vibrational spectroscopy, evidencing the limitations of the CO-based coatings in comparison with MMWC, which has a higher viscosity and better adhesion properties. The usage of MMWC holds promise for poplar wood protection, with potential industrial applications. Keywords: chitosan composites; FTIR; natural protectors; poplar wood; white-rot fungus 35 1. Introduction In the field of wood protection, significant efforts have been devoted over the last decades to the assessment of natural products that can pose an alternative to other traditionally used chemical compounds, which have toxic effects on human beings and the environment [1]. In this sense, renewable polymers have attracted intense industrial interest, and, amongst them, chitosan has 40 particularly promising application perspectives [2]. This natural polysaccharide derived from chitin is the second most abundant polymer in nature. Chitosan consists of chains of N-acetyl-Dglucosamine and D-glucosamine, and features a cationic character, which confers unique properties on it [3]. Among its main characteristics, such as its bioactivity, non-toxicity or biodegradability, its antimicrobial effect is especially relevant [4–6]. Its activity against different fungi, gram positive and 45 Coatings 2018, 8, x FOR PEER REVIEW 2 of 15 gram negative bacteria, is ascribed to the positive charge of amino group (NH3+), which interacts electrostatically with the surface of the cellular membrane, destabilizing it. As a consequence, the presence of chitosan inside the cell can lead to intracellular responses such as inactivation or blocking of enzymes activities, and of DNA transcription and translation [7,8]. The antifungal activity of chitosan not only depends on the fungus species, but also on its 50 molecular weight (MW), polymerization degree (PD) or deacetylation degree (DD). For instance, chito-oligosaccharides with low MW, PD and DD have been reported to be more effective on phytopathogenic fungi than chitosan with higher MW, PD or DD [9–11]. Another known advantage from chitosan and its oligomers is associated with their sorption and chelating properties. The cationic nature of the polymer allows it to bind to different chemicals 55 species, ranging from organic macromolecules to metal nanoparticles [12–14]. Natural oils or extracts of natural products can be incorporated into the chitosan matrix, e.g., by cross-linking, producing solutions, films or beads [15–17]. In the particular case of propolis, its polyphenols have been reported to form hydrogen bonds and covalent bonds with the functional groups in chitosan [18]. This results in a stabilization of the propolis components in the polymer and in an enhancement of the 60 antimicrobial activity due to a synergistic behavior [19,20]. In the case of nanometals, e.g., silver nanoparticles, chitosan and its derivatives can also be used in their synthesis, provided that chitosan can act as reducer as well as a stabilizer : the polymer matrix facilitates the nanoparticles preparation in one-step process, resulting in nanosilver with an ideal particle size [21]. Further, the resulting chitosan-nanosilver composites present a higher antimicrobial effect [22]. 65 Chitosan-based composites have been successfully applied to the preservation of wood against mold fungi, as well as brown-rot and white-rot fungi [23–26], both in vitro and over beech and fir [27], Monterrey pine [28], Scot pine [29] and other hardwood and softwood species. Nonetheless, to the best of the authors’ knowledge, to date no studies have focused on chitosan applications in order to protect poplar species. Populus spp., belonging to the Salicaceae family, are one of the most cultivated 70 woody plants for industrial purposes. Its wood is one of the least expensive hardwoods and, although rarely used in the production of fine furniture, it is extensively used for pulp, panel productions and many other commercial applications [30,31]. These fast-growing trees therefore have a large economic impact worldwide, together with a significant importance from an environmental perspective [32]. According to EN 350:2016 rule, poplar wood is a non-durable species [33], and some 75 studies have evidenced that it is highly susceptible to Trametes versicolor [34,35]. Therefore, chitosanbased composites can be applied as a potential tool to avoid the white-rot decay of Populus spp. wood, improving its natural durability and increasing its technological uses. The first objective of the study was to investigate the in vitro antifungal activity against T. versicolor of two different molecular weight chitosan treatments: medium molecular weight chitosan 80 (MMWC) and chitosan oligomers (CO); the latter –which resulted in a better minimum inhibitory concentration (MIC) value– was also tested in binary and ternary combinations with propolis (P) and nanosilver (nAg). In a second stage, once the MIC values had been determined, the best treatments were evaluated on poplar wood blocks as surface protectors to prevent wood decay, with a view to their practical application. Wood biodegradation was monitored by microscopy and by tracking 85 chemical alterations. For this latter purpose, Fourier-transform infrared spectroscopy (FTIR) technique was chosen, provided that it is quickly consolidating as an easy-technique to characterize and evaluate the decay of wood [36–38]. 2. Materials and methods 2.1. Reagents, fungal isolate and wood 90 Medium molecular weight chitosan (CAS No. 9012-76-4), with 60-130 kDa and 90% deacetylation, was acquired from Hangzhou Simit Chemical Technology Co. (Hangzhou, China). Propolis, with a content of ca. 10% w/v of polyphenols and flavonoids, came from the Duero river basin region (Burgos, Spain). Silver nitrate (CAS number 7761-88-8) was supplied by Merck Millipore Coatings 2018, 8, x FOR PEER REVIEW 3 of 15 (Darmstadt, Germany). All the other reagents used to synthetize or prepare the solutions were of 95 analytical grade. The isolate of T. versicolor (L.) Lloyd 1920 (DSM 3086 strain, CECT 20804) was supplied by the Spanish Type Culture Collection (Valencia, Spain) and was cultivated on potato dextrose agar (PDA) (supplied by Scharlau, Barcelona, Spain) for all assays. The wood blocks were supplied by the Wood Technology Laboratory at Universidad de 100 Valladolid from P. euroamericana I-214 clone. 2.2. Chitosan-based solutions The solutions of MMWC, CO, P, nAg and the CO-based binary and ternary mixtures -tested in vitro for the inhibition of T. versicolor mycelial growth and as wood coatingswere prepared starting from commercial medium MW chitosan, which was dissolved in acetic acid (1 %) under constant 105 stirring at 60 °C for 2 h. The pH value was then adjusted from 4.5 to 6.5 with potassium methoxide. Subsequently, oligo-chitosan was prepared by oxidative degradation of the solubilized MMWC by addition of hydrogen peroxide (0.3 M), obtaining a MW of 2 kDa [39]. Propolis constituents soluble in ethanol were extracted by grinding the resin and adding it to a hydroalcoholic solution (ethanol 30%), followed by stirring for 72 h at room temperature, and by filtration with a stainless steel 220 110 mesh to remove insoluble particles [40]. Silver nanoparticles were synthetized by a sonication method: 50 mL of silver nitrate (50 mM) were first mixed with 50 mL of sodium citrate (30 mM) as a reducing agent, and the solution was heated up to 90 °C until it turned from colorless to pale yellow, which then became more intense. The yellowish solution was sonicated for 3-5 minutes and allowed to stabilize for at least 24 h in a refrigerator at 5 °C [41]. 115 The binary and ternary solutions were prepared according to Martín-Gil et al. [42], by mixing of the solutions described above at the desired concentrations (Table 1), followed by sonication for 1 min with a probe-type UIP1000hdT ultrasonicator (Hielscher, Teltow, Germany; 1000 W, 20 kHz). Table 1. Concentrations used in the in vitro experiments (MMWC, COs and P are given in mg·mL-1, and that of nAg in µg·mL-1). 120 Treatment Concentration Control 0.0 MMWC 1.0 2.0 4.0 7.0 10.0 CO 1.0 1.5 2.0 2.5 3.0 P 0.1 0.2 0.3 0.4 0.5 nAg 1.0 1.5 2.0 2.5 3.0 CO-P 0.5 – 0.05 1.0 – 0.1 2.0 – 0.2 CO-nAg 0.5 – 0.5 1.0 – 1.0 2.0 – 2.0 P-nAg 0.05 – 0.5 0.1 – 1.0 0.2 – 2.0 CO-P-nAg 0.5 – 0.05 – 0.5 1.0 – 0.1 – 1.0 2.0 – 0.2 – 2.0 2.3. In vitro assays Each solution was incorporated into PDA at a ratio of 1:10 (v/v). PDA had been sterilized for 20 min at 121 °C, and had been cooled down to 50 °C before the addition of the treatments (or of distillated and sterilized water in the case of control). 20 mL of the medium were then spread in Petri dishes and, once they had solidified, 5 mm in diameter discs of young mycelia of T. versicolor were 125 placed on the center of each Petri dish, followed by incubation in the dark at 25 °C and 65% HR. Three replicates were performed for each treatment. Finally, radial growth was measured on a daily basis until the mycelium reached the edges of the control Petri dish (8 days), and the inhibition growth percentage was calculated. 2.4. Wood coating assays 130 Coatings 2018, 8, x FOR PEER REVIEW 4 of 15 On the basis of the in vitro study results, the best treatments (either individual, binary or ternary mixtures) were assayed as a coating over poplar wood in order to prevent its degradation against T. versicolor fungus. Small poplar wood blocks (5×5×20 mm), previously dried at 103±2 °C to determinate their initial dry weight, were soaked in the different solutions: water (control), MMWC, CO, CO-P and CO-P135 nAg (36 blocks per treatment) for 12 h at room temperature. A concentration of 20 mg·mL-1 for MMWC, 20 mg·mL-1 for CO, 2 mg·mL-1 for P, and 20 µg·mL-1 for nAg were used. These doses, which were ten times higher than the obtained MIC values from binary and ternary mixtures, were chosen on the basis of adsorption, penetration and fixation tests, so as to compensate for the influence of the dipping time on the uptake of the preservative solution and to minimize wood swelling, as reported 140 by Eikenes et al. [29] and Larnoy et al. [43]. Excess of liquid was then removed and the wood blocks were dried in a chamber for 24 h at 45 °C. The coated blocks were placed on Petri dishes -six per platenewly covered until the edges by T. versicolor mycelia, which were again incubated at 27 °C and 70% of relative humidity (RH) for 30 days. One Petri dish, with six blocks, of each treatment was removed every 5 days for analysis. The 145 mycelia on the wood surface were carefully cleaned, the small blocks were then dried at 103±2 °C to quantify their dry weight after fungal attack, and the mass loss was determined by subtracting it from the initial weight according to EN 113:1996 [44]. 2.5. Microscopy and FTIR studies In order to monitor wood degradation, a Leica (Wetzlar, Germany) microtome was used to cut 150 thin wood sections (30 µm), which were analyzed by optical microscopy (with a Leica DMLM Transmission Optical Microscope). Thin cut sections were also grinded and mixed with KBr (in 1:300 ratio) to prepare pellets to study the FTIR spectrum by direct transmittance technique, using a Thermo Scientific (Waltham, MA, USA) Nicolet iS50 FTIR spectrometer. The spectra were collected in the 400-4000 cm-1 region at room temperature with a 1 cm-1 spectral resolution; a total of 64 scans 155 were co-added and the resulting interferogram was averaged. The obtained spectra were corrected using the advanced ATR correction algorithm available in OMNIC software suite (Thermo Scientific), and were normalize using the 1048 cm-1 band, assigned to CO stretching, in agreement with Colom et al. [36]. 2.6. Statistical analyses 160 All the statistical analyses were performed using R software (v. 3.4.4) (R Development Core Team, 2018). A total of 216 data, corresponding to 15 different individual groups (6 samples × 3 preservative agents × 5 concentration levels), 1 control group (6 samples), 3 binary groups (6 samples × 3 groups × 3 concentration levels) and 1 ternary group (6 samples × 3 levels) were analyzed. Prior to the analyses, the assumptions of independence, normality and homoscedasticity were checked for 165 all groups. Since the data structure did not meet the normality requirement, checked with ShapiroWilks test, or the homoscedasticity requirement, checked with Bartlett’s test, the usual comparative analysis (ANOVA) could not be used. Bootstrapping, Welch’s test and robust homogenous groups were used instead. 3. Results and discussion 170 3.1. Minimum inhibitory concentration The antifungal activity of the chitosan-based composites against T. versicolor was evaluated in a typical in vitro inhibition growth experiment. The results, collected after seven days of incubation, are shown in Table 2. MMWC presented a moderate activity against mycelial growth, with significant differences in comparison with the control treatment. However, low doses resulted in a low inhibition 175 and higher concentrations (up to 10 mg·mL-1) were needed to approach 100% mycelial growth inhibition. In contrast, CO exhibited a high inhibitory activity even at the lowest doses; e.g., 1 mg·mL1 of CO inhibited 79% of mycelial growth. As expected, the increase in CO concentration resulted in Coatings 2018, 8, x FOR PEER REVIEW 5 of 15 an increase of T. versicolor mycelial growth inhibition, attaining 100% inhibition (MIC) at a concentration of 3 mg·mL-1. 180 As regards the differences observed between the activities of MMWC and CO, the results obtained in this study would be in agreement with other in vitro assays reported in the literature, such as those mentioned in the introduction or those by Matei et al. [41] and Rahman et al. [9], who found a better antifungal activity for low MW (less than 20 kDa) chitosan than for medium or high MW (more than 140 kDa) chitosan. Nonetheless, it should be clarified that this behavior may not be 185 extrapolated to all fungi: for instance, Younes et al. [10] found that fungal growth decreased with increasing MW for Fusarium oxysporum, thus pointing at the need to evaluate the optimum MW for each species. In relation to the MIC, Stössel and Leuba [45] reported that –for native chitosan– MIC values could vary from 0.001 to 2.5 mg·mL-1, depending on the phytopathogenic fungus. According to Rabea 190 et al. [46], the MIC value depends on factors such as the type of chitosan, the fungus under study, the chemical or nutrient composition of the substrate and the environmental conditions. Another work on Sphaeropsis sapinea and Trichoderma harzianum wood-degrading fungi suggested that the application of 1 mg·mL-1 of low-molecular weight chitosan reduced growth rate by a factor of 3 with respect to the control [47], in line with the results reported by Silva-Castro et al. [40], who found that 195 1 mg·mL-1 of CO inhibited 86% of the mycelial growth of Heterobasidium annosum basidiomycete fungus. Thus, in view of aforementioned MIC values, it may be inferred that T. versicolor would show an intermediate sensitivity (i.e., would be moderately sensitive) to chitosan/CO alone, albeit such resistance would not be as high as that of, for example, Macrophomina phaseolina (for which MIC values of water-soluble chitosan as high as 12.5 mg·mL-1 were found [48]). Consequently, it seemed 200 necessary to explore binary or ternary combinations with other compounds with antifungal activity in order to search for synergies and to attain lower MIC values. Propolis exhibited a higher antifungal activity against T. versicolor than CO: in propolis-based treatments, 85% growth inhibition was attained with 0.1 mg·mL-1, with a MIC value of 0.5 mg·mL-1 (six times lower than MIC of CO). The use of propolis as an antifungal agent is well-known, and its 205 effectiveness has been proved against Candida spp. and other human pathogens [49–51]. This has promoted its use in the production of films to control foodborne fungi [52,53]. With regard to phytopathogenic oomycetes (Phytophthora alni and Phytophthora plurivora), 0.1 mg·mL-1 of propolis ethanolic extract resulted in 100% inhibition [40]. Although there is no data concerning propolis action against white-rot fungi (a thorough search of the relevant literature did not yield any reports 210 on the antifungal activity of propolis ethanolic extracts to control T. versicolor growth), other natural products were assessed by Zhang et al. [54], who evaluated the activity against white-rot fungi of 41 monoterpenes (which are part of the composition of propolis). They found that carvacrol at a concentration of 0.4 mg·mL-1 led to 100% growth inhibition against Trametes hirsuta, Schizphylhls commune, and Pycnoporus sanguineus, i.e., its MIC value was similar to that reported herein for P. 215 Regarding nAg treatments, just 1 µg·mL-1 led to 85% mycelial growth inhibition, which increased up to 100% at a concentration of 3 µg·mL-1 (MIC value). It should be clarified that the remarkable ability of nanosilver to inhibit fungal growth depends not only on the fungal species, but on the nanoparticles characteristics -shape, size, synthesis method, stabilization, etc.- [55], which makes direct comparisons difficult. For instance, a study was carried out to test three types of nanosilver 220 against eighteen plant pathogenic fungi, and while no variation was detected at relatively low doses (10 µg·mL-1), at higher concentrations (100 µg·mL-1) the maximum inhibition for most fungi was shown by dark brown colloid nanoparticles [56]. Apropos of wood-degrading fungi, silver nanoparticles synthesized using turnip leaf extract were assayed against four fungi, showing slight to moderate growth inhibition (with 2 and 4 mg of nanosilver on paper discs) [57]. Although in both 225 studies the nanoparticles presented a reasonably good activity, the MIC value found in the present study indicates that T. versicolor would be very sensitive to nAg. Since the antifungal activity of CO was noticeably higher than that of MMWC, only composites of CO, P and nAg, in binary and ternary mixtures, were assessed. These composites presented significant differences in comparison with the same concentrations of individual products. The CO230 Coatings 2018, 8, x FOR PEER REVIEW 6 of 15 P binary composites inhibited 100% of mycelial growth with 2 and 0.2 mg·mL-1, respectively (vs. MIC values of 3 mg·mL-1 and 0.5 mg·mL-1 for individual treatments with CO and P, respectively), suggesting that the mixture of both natural products would enhance the antifungal activity against T. versicolor. A similar behavior has also been reported by other authors, who observed that the addition of propolis to chitosan films enhanced their in vitro antimicrobial effect [18]. However, it is 235 worth noting that this synergistic behavior cannot be extrapolated to all pathogens, provided that Mascheroni et al. [58] reported disparate results for different foodborne fungi, with lower MICs for propolis-only treatments than for chitosan-propolis beads in some cases. Likewise, Silva-Castro et al. [40] found no statistically significant differences between CO-P composites and propolis-only treatments against P. alni and P. plurivora. 240 Apropos of the binary CO-nAg composite, it resulted in 100% growth inhibition (MIC value) for a concentration of 2 mg·mL-1 and 2 µg·mL-1, respectively (lower than the MICs of the corresponding individual products too). However, a different behavior was observed at lower doses of the mixture, provided that significant differences between CO-nAg and nAg-only treatments were not found at 0.1 µg·mL-1, suggesting that the nAg dose in the composite would be crucial (as noted above, T. 245 versicolor showed a much higher sensitivity to nAg than to chitosan). The results at a higher concentration would be in good agreement with other studies in which it has been demonstrated that the composites of both products have a greater antifungal activity [59], while the behavior found at lower nAg doses would be in line with the findings of Velmurugan et al. [60], who reported that chitosan with silver nano-sized particles exhibited a similar antifungal effect than the nanoparticles 250 alone against wood staining fungi, given the remarkable antifungal activity of the latter. The third binary mixture, P-nAg, presented a MIC value of 0.2 mg·mL-1 and 2 µg·mL-1 for P and nAg, respectively, which was also lower than MICs found for the treatments based on the individual products. However, at lower concentrations (0.1 mg·mL-1 and 0.1 µg·mL-1) of P-nAg, again there were not significant differences in comparison with the nAg individual solutions, resulting in similar 255 inhibition percentages (around 85%). This would reinforce the interpretation discussed above, in which nAg would play a key role in T. versicolor inhibition, given the high sensitivity of the fungus to this component. The dependence of the behavior on the particular pathogen is evidenced by the different results reported for the control of other forest pathogens: Silva-Castro et al. [40]found that P-nAg composites showed a similar effect to that of CO against Fusarium circinatum, a lower effect 260 than that of CO against Diplodia pinea, and an enhanced behavior vs. CO-only treatments for the control of Phytophthora cambivora. Finally, the ternary mixture reached full mycelial growth inhibition at a MIC value of 2 mg·mL1 of CO, 0.2 mg·mL-1 of P and 0.2 µg mL-1 of nAg, so it would not present an advantageous behavior as compared to the binary composites. This same CO-P-nAg ternary composite has been tested 265 against Diplodia seriata [41], Bipolaris oryzae [61] and eight forest phytopathogenic fungi [40], attaining a higher inhibition than the individual or binary products at concentrations of 20–25 mg·mL-1, but leading to a similar effect than that of binary solutions at lower concentrations, as it happens in the present study. Paradoxically, at the lowest dose assayed herein (0.5 mg·mL-1 of CO, 0.05 mg·mL-1 of P and 0.5 µg·mL-1 of nAg), CO-P-nAg showed a noticeable higher antifungal effect against T. versicolor 270 (79% inhibition) than the binary mixtures (51%, 64% and 47% inhibition for CO-P, CO-nAg and PnAg, respectively). Table 2. Effect of treatments concentration on the mycelial growth of T. versicolor (in vitro) seven days after incubation. The concentrations of MMWC, COs and P are given in mg·mL-1, and that of nAg in µg·mL-1. 275 Treatment Concentration Inhibition percentage (%) Mean - Confidence Interval and homogeneous groups Shapiro Wilk p-value Levene's test p-value Welch’s test p-value Control 0.0 0.000  0.000 C 0.000 -- -- MMWC 1.0 22.708  2.027 a 0.324 0.08629 0.0000 2.0 42.083  1.429 b 0.505 4.0 75.375  1.970 e 0.377 Coatings 2018, 8, x FOR PEER REVIEW 7 of 15 7.0 90.250  1.846 i 0.988 10.0 95.542  1.331 j k 0.001 CO 1.0 78.750  1.094 f 0.110 0.0417 0.0000 1.5 87.708  0.857 h 0.035 2.0 92.333  0.991 i j 0.433 2.5 97.000  0.992 k 0.680 3.0 99.997  0.004 l 0.001 P 0.1 84.541  1.135 g 0.836 0.0207 0.0000 0.2 88.333  1.173 i 0.911 0.3 93.333  1.148 j 0.850 0.4 97.500  0.909 k 0.421 0.5 99.997  0.004 l 0.001 nAg 1.0 85.000  1.270 g 0.960 0.0214 0.0000 1.5 91.333  1.221 h 0.843 2.0 94.217  1.177 j 0.473 2.5 97.333  0.753 k 0.725 3.0 99.997  0.004 l 0.001 CO-P 0.5 – 0.05 51.458  2.472 c 0.540 0.0037 0.0000 1.0 – 0.1 89.167  0.451 h 0.001 2.0 – 0.2 99.997  0.003 l 0.001 CO-nAg 0.5 – 0.5 64.583  0.427 d 0.001 0.0281 0.0000 1.0 – 1.0 84.583  0.740 g 0.091 2.0 – 2.0 99.996  0.003 l 0.001 P-nAg 0.05 – 0.5 47.292  1.673 c 0.212 0.1652 0.0000 0.1 – 1.0 84.167  0.639 g 0.001 0.2 – 2.0 99.997  0.004 l 0.001 CO-P-nAg 0.5 – 0.05 – 0.5 77.500  1.081 f 0.110 0.0190 0.0000 1.0 – 0.1 – 1.0 85.208  1.348 g 0.804 2.0 – 0.2 – 2.0 99.997  0.004 l 0.001 3.2. Wood preservation assays 3.2.1. Weight loss Surface treatments with the chitosan-based composites were applied to mini-blocks of poplar wood in order to prevent white-rot decay. Weight loss was recorded every 5 days (see Table 3). The 280 non-treated mini-blocks (control) and those treated with chitosan oligomers-based treatments (CO, CO-P and CO-P-nAg) started losing weight from second sampling (10 days), in contrast with those treated with MMWC. In the third sampling (after 15 days), degradation caused by T. versicolor increased for all treatments, although with significant differences among the control and all the other treatments. Non-covered blocks exhibited a weight loss of up to 27.3 %, followed by those treated 285 with CO-P-nAg (22.8%), CO-P (14.7%), CO (7%) and MMWC (3%). The differences between coated and non-coated samples further increased in the fourth sampling (after 20 days), in which the weight loss only increased slightly for the treated blocks, while degradation reached 32.9% for the control. 25 days after the exposure to fungus, the wood treated with MMWC still exhibited the lowest weight loss (7.7%), while weight loss for the CO-based treatments increased, albeit still with significant 290 differences as compared to the control (39.8 %). In the last sampling (after 30 days), the wood blocks treated with the ternary composite reached a similar degradation level to that of the control -without significant differences-, in contrast with MMWC-treated samples, for which the weight loss (10.8%) remained well-below that of the control (42.3%). Table 3. Effect of coating treatments and time on weight loss for Populus spp. wood-blocks exposed 295 to white-rot fungus T. versicolor. Treatment Time (days) Weight loss (g) Shapiro Wilk Levene’s test Welch’s test Coatings 2018, 8, x FOR PEER REVIEW 8 of 15 Mean - Confidence Interval and homogeneous groups p-value p-value p-value Control 5 0.138  0.155 a 0.004 0.1973 0.0000 10 5.352  1.315 bc 0.079 15 27.261  3.037 ij 0.127 20 32.888  4.233 jk 0.073 25 39.774  2.036 l 0.949 30 42.353  1.866 l 0.470 MMWC 5 0.000  0.000 a 0.000 0.1132 0.0000 10 0.000  0.000 a 0.000 15 3.478  0.541 b 0.534 20 7.177  0.570 c 0.900 25 10.157  1.095 d 0.955 30 11.455  1.171 d 0.412 CO 5 0.000  0.000 a 0.000 0.0001 0.0000 10 4.311  0.883 b 0.825 15 6.570  1.226 c 0.759 20 13.524  0.937 e 0.999 25 22.190  0.890 h 0.961 30 26.871  1.164 i 0.373 CO-P 5 0.000  0.000 a 0.000 0.0254 0.0000 10 7.567  0.889 c 0.610 15 13.578  1.084 e 0.996 20 17.276  1.154 f 0.869 25 29.662  1.174 j 0.340 30 32.856  1.039 k 0.289 CO-P-Ag 5 0.000  0.000 a 0.000 0.0342 0.0000 10 10.615  1.071 d 0.138 15 20.150  1.185 g 0.660 20 26.864  1.154 i 0.971 25 35.074  1.341 k 0.762 30 39.943  2.357 l 0.882 The poplar wood mini-blocks from the control treatment presented a higher degradation rate than those of beech wood exposed to T. versicolor in the study carried out by Mohebby [38]: in the latter, a weight loss of ca. 20% was recorded on the 28th day of the experiment, half of the one recorded 300 in this study after 30 days (42%). This can be readily ascribed to the high susceptibility of Populus spp. wood to this fungus [32,34]. With regard to the differences in the weight loss rates for the treated samples, although most treatments resulted in a slower degradation than that of the control, MMWC was clearly the most effective protective agent (Figure 1). This should be ascribed to its higher viscosity and better 305 adhesive properties, adhesion is directly related to the DD of chitosan: if chitosan DD increases, the number of positive charges also increases, which leads to improved adhesive properties [62], which would result in a better coating on the surface of wood blocks. This would be in agreement with Eikenes et al. [29], who demonstrated that medium MW chitosan (at a concentration of 50 mg·mL-1) was able to avoid the decay caused by brown-rot fungi on Scots pine, performing better than low 310 MW chitosan, as it also happened in this study. Low doses of this type of chitosan (lower than 10 mg·mL-1) were also found to be effective against white-rot fungi, according to Nowrouzi et al. [27]. Of course, the wood preservation effect would largely depend on the concentration of chitosan, as demonstrated by El-gamal et al. [23] in relation to the growth of fungi on treated wood samples from historic artifacts (fungal growth decreased with the increase in chitosan concentration). 315 Concerning the effectiveness as wood protecting agents of the composites, it was lower than that of the chitosan-only treatments (especially in comparison with MMWC), in contrast with the results from the in vitro assays. This unexpected result may be explained by their low viscosity and poor adhesion properties [63], which may be enhanced by adding thickening agents to chitosan oligomers, such as natural gums, starches, pectins, alginate and carrageenan [64]; by preparing CO–clay based 320 Coatings 2018, 8, x FOR PEER REVIEW 9 of 15 composite materials [65]; or by using other impregnation procedures (involving vacuum and pressure). In view of aforementioned results, MMWC would be the preferred choice for industrial applications, not only due to its higher effectiveness, but also because of its fast and facile preparation process. 325 Figure 1. Poplar wood samples 30 days after exposure to T. versicolor: (a) control wood sample and (b) sample treated with medium molecular weight chitosan. 3.2.2. Wood degradation monitoring The decay of the wood-blocks was tracked using microscopy techniques. In the control sample, 330 changes such as cavities in the cell wall and a quick development of fungal hyphae were easily recognized, the change between undecayed control wood and the last sampling (after 30 days) can be observed by comparing Figure 2a and Figure 2b, respectively. The hyphae progressively increased over the 30 days, resulting in a strong structure interconnected with the wood one, as well as in holes in the cell walls and in a general degradation of the vessels and fibers of the poplar wood, confirming 335 the high susceptibility of Populus spp. wood to T. versicolor [32]. Analogous effects were identified with SEM microscopy on uncovered blocks of the softwood from Paulownia fortune attacked by T. versicolor [66], providing evidence that the fungus can tunnel along the cellulose microfibrils, resulting in the formation of holes in transverse sections. Changes in the samples impregnated with MMWC were much less evident, even 30 days after exposure, with an associated weigh loss of 10% 340 (Figure 2c). Figure 2. Optical micrographs (at 500× magnification) of the surface of poplar wood samples: (a) undecayed control wood sample prior to exposure to T. versicolor; (b) control wood sample 30 days after exposure to T. versicolor; and (c) sample treated with medium molecular weight chitosan 30 days 345 after exposure to T. versicolor. FTIR spectroscopy was also used to follow the decay –by tracking chemical changes– of the poplar wood mini-blocks. The infrared spectrum of undecayed control wood (Figure 3 ) was almost