Elicitation test of cell suspensions using as elicitor an extract of "Moringa oleifera" leaves

Traballo fin de grao (UDC.CIE). Bioloxía. Curso 2016/2017[Resumen] En este trabajo de investigación se estudió el efecto de extractos de hojas de Moringa sobre la producción de compuestos fenólicos solubles en el medio extracelular de suspensiones celulares de Capsicum annuum var. annuum. Asimismo, se analizó el efecto de estos extractos sobre la capacidad antioxidante de las suspensiones celulares. Los resultados indicaron que altas concentraciones del extracto metanólico de hojas de Moringa provocó un incremento significativo sobre la producción de fenoles solubles en el medio extracelular de las suspensiones celulares de C. annuum. Sin embargo, este incremento no se vió acompañado con un aumento en la actividad antioxidante. En este trabajo de investigación es la primera vez que se comprueba el efecto elicitor de extracto de Moringa sobre suspensiones celulares vegetales, y abre una prometedora puerta sobre el uso de extractos para incrementar la bioproducción de compuestos fenólicos.[Abstract] This research work focuses on the study of the effect of Moringa leaf extracts on the production of phenolic compounds that are soluble in the extracellular medium of cell suspensions of the plant Capsicum annuum var. annuum. Likewise, the effect of these extracts on the antioxidant capacity of the cell suspensions was analysed. The results show that high concentrations of the methanolic extract of Moringa leaves increased significantly the production of soluble phenols in the extracellular medium of C. annuum cell suspensions. However, this increase was not followed by an increase in the antioxidant activity. This research work was the first time that the effect that the Moringa extracts elicitor has over plant cell suspensions has been tested, and it opens a promising door on the use of extracts to increase the bioproduction of phenolic compounds

Deciphering the Effect of Light Wavelengths in Monilinia spp. DHN-Melanin Production and Their Interplay with ROS Metabolism in M. fructicola

Pathogenic fungi are influenced by many biotic and abiotic factors. Among them, light is a source of information for fungi and also a stress factor that triggers multiple biological responses, including the activation of secondary metabolites, such as the production of melanin pigments. In this study, we analyzed the melanin-like production in in vitro conditions, as well as the expression of all biosynthetic and regulatory genes of the DHN–melanin pathway in the three main Monilinia species upon exposure to light conditions (white, black, blue, red, and far-red wavelengths). On the other hand, we analyzed, for the first time, the metabolism related to ROS in M. fructicola, through the production of hydrogen peroxide (H2O2) and the expression of stress-related genes under different light conditions. In general, the results indicated a clear importance of black light on melanin production and expression in M. laxa and M. fructicola, but not in M. fructigena. Regarding ROS-related metabolism in M. fructicola, blue light highlighted by inhibiting the expression of many antioxidant genes. Overall, it represents a global description of the effect of light on the regulation of two important secondary mechanisms, essential for the adaptation of the fungus to the environment and its survival.This research was funded by the national projects AGL2017-84389-C2-1-R and PID2020-1157AGL2017 from the Ministry of Science, Innovation and Universities (MCIU, Spain) and from the CERCA Program/Generalitat de Catalunya. L. Verde-Yáñez received a Ph.D. fellowship PRE2018-085428 from Agencia Estatal de Investigación (AEI, Spain).info:eu-repo/semantics/publishedVersio

Influencia de la luz en el crecimiento y desarrollo de Monilinia spp. y su efecto en la producción y biosíntesis de melanina

La podridura marró és una malaltia causada pel fong Monilinia spp. en fruita de pinyol i de llavor, provocant grans pèrdues econòmiques, especialment durant el període postcollita. Estudis realitzats els darrers anys han assenyalat la influència de factors ambientals com la humitat i la temperatura en el desenvolupament de la malaltia, encara que poc s'ha descrit sobre la llum. Per això, en aquesta tesi es va estudiar la interacció entre el patogen Monilinia spp. (M. laxa, M. fructicola i M. fructigena) i el factor ambiental llum, com a longitud d'ona, en el creixement i desenvolupament de Monilinia spp., així com la seva resposta associada a la biosíntesi de la melanina. Des del punt de vista de Monilinia spp., es va avaluar l'efecte de les longituds d'ona sobre el desenvolupament in vitro, la regulació de gens de morfogènesi i la capacitat d'infectar nectarines. Aquest estudi va revelar una plasticitat fenotípica alta per part de M. laxa i M. fructicola en resposta a les longituds d'ona, mentre que M. fructigena no va mostrar grans canvis (Capítol 1). Posteriorment, es va estudiar la melanina a Monilinia spp. Per això, es va quantificar el pigment tipus melanina mitjançant espectrofotometria, es va identificar i analitzar l'expressió dels gens de la ruta DHN tant en condicions in vitro com en nectarines, primer en condicions de foscor (Capítol 2), i posteriorment a diferents longituds d'ona (Capítol 3). Els resultats, en condicions in vitro, van mostrar que M. fructigena i M. fructicola van produir més quantitat de melanina que M. laxa. En nectarines, aquestes dues mateixes espècies van incrementar la producció de melanina a mesura que es desenvolupava la malaltia, mentre que M. laxa va produir més melanina a l'inici de la malaltia. A més, es va identificar la presència dels gens implicats a la ruta DHN al genoma de Monilinia spp. (Capítol 2) i se'n va demostrar la dependència de la llum (Capítol 3). A totes les espècies va destacar la producció del pigment tipus melanina sota la llum negra i/o sota la llum blava. D'altra banda, en M. fructicola es va observar que el metabolisme de les ROS estava estretament relacionat amb la melanina i influenciat per la llum (Capítol 3). Finalment, es va investigar la ruta L-DOPA en Monilinia spp. Les anàlisis van permetre identificar els pigments derivats d'aquesta ruta, feomelanina i eumelanina, garantint el creixement i el desenvolupament de Monilinia spp.La podredumbre parda es una enfermedad causada por el hongo Monilinia spp. en fruta de hueso y de pepita, provocando grandes pérdidas económicas, especialmente durante el periodo postcosecha. Estudios realizados en los últimos años han señalado la influencia de factores ambientales como la humedad y la temperatura en el desarrollo de la enfermedad, aunque poco se ha descrito sobre la luz. Por ello, en la presente tesis se estudió la interacción entre el patógeno Monilinia spp. (M. laxa, M. fructicola y M. fructigena) y el factor ambiental luz, como longitud de onda, en el crecimiento y desarrollo de Monilinia spp., así como sus respuestas asociadas a la biosíntesis de la melanina. Desde el punto de vista de Monilinia spp., se evaluó el efecto de las longitudes de onda sobre su desarrollo in vitro, la regulación de genes de morfogénesis y su capacidad de infectar nectarinas. Este estudio reveló una alta plasticidad fenotípica por parte de M. laxa y M. fructicola en respuesta a las longitudes de onda, mientras que M. fructigena no mostró grandes cambios (Capítulo 1). Posteriormente, se estudió la melanina en Monilinia spp. Para ello, se cuantificó los pigmentos tipo melanina mediante espectrofotometría, se identificó y analizó la expresión de los genes de la ruta DHN tanto en condiciones in vitro como en nectarinas, primero en condiciones de oscuridad (Capítulo 2), y posteriormente a diferentes longitudes de onda (Capítulo 3). Los resultados, en condiciones in vitro, mostraron que M. fructigena y M. fructicola produjeron mayor cantidad de melanina que M. laxa. En nectarinas, estas dos mismas especies incrementaron la producción de melanina a medida que se desarrollaba la enfermedad, mientras que M. laxa produjo más melanina al inicio de la enfermedad. Además, se identificó la presencia de los genes implicados en la ruta DHN en el genoma de Monilinia spp. (Capítulo 2) y se demostró su dependencia de la luz (Capítulo 3). En todas las especies destacó la producción de los pigmentos tipo melanina bajo la luz negra y/o bajo la luz azul. Por otro lado, en M. fructicola se observó que el metabolismo de las ROS estaba estrechamente relacionado con la melanina e influenciado por la luz (Capítulo 3). Finalmente, se investigó la ruta L-DOPA en Monilinia spp. Los análisis permitieron identificar los pigmentos derivados de esta ruta, feomelanina y eumelanina, garantizando el crecimiento y desarrollo de Monilinia spp.Brown rot is a disease caused by the fungus Monilinia spp. in stone and pome fruit, causing great economic losses, especially during the postharvest period. Studies carried out in recent years have pointed out the influence of environmental factors such as humidity and temperature on the development of the disease, although little has been described about light. Hence, in this thesis was studied the interaction between the pathogen Monilinia spp. (M. laxa, M. fructicola and M. fructigena) and the environmental factor light, such as wavelength, in the growth and development of Monilinia spp., as well as its responses associated with melanin biosynthesis. From the point of view of Monilinia spp., was evaluated the effect of light wavelengths on its in vitro development, the regulation of morphogenesis genes and its ability to infect nectarines. This study revealed high phenotypic plasticity by M. laxa and M. fructicola in response to light wavelengths, while M. fructigena did not show large changes (Chapter 1). Subsequently, melanin was studied in Monilinia spp. To do this, the melanin-like pigments was quantified by spectrophotometry, the expression of the DHN pathway genes was identified and analyzed both in vitro conditions and in nectarines, first in darkness conditions (Chapter 2), and subsequently at different wavelengths (Chapter 3). The results, under in vitro conditions, showed that M. fructigena and M. fructicola produced a greater amount of melanin than M. laxa. In nectarines, these same two species increased melanin production as the disease developed, while M. laxa produced more melanin at the beginning of the disease. Furthermore, the presence of the genes involved in the DHN pathway was identified in the genome of Monilinia spp. (Chapter 2) and its dependence on light was demonstrated (Chapter 3). In all species highlighted the production of melanin-like pigments under black light and/or blue light. On the other hand, in M. fructicola was observed that ROS metabolism was closely related to melanin and influenced by light (Chapter 3). Finally, the L-DOPA pathway was investigated in Monilinia spp. The analyzes allowed the identification of the pigments derived from this pathway, pheomelanin and eumelanin, guaranteeing the growth and development of Monilinia spp

Deciphering the Effect of Light Wavelengths in <i>Monilinia</i> spp. DHN-Melanin Production and Their Interplay with ROS Metabolism in <i>M. fructicola</i>

Pathogenic fungi are influenced by many biotic and abiotic factors. Among them, light is a source of information for fungi and also a stress factor that triggers multiple biological responses, including the activation of secondary metabolites, such as the production of melanin pigments. In this study, we analyzed the melanin-like production in in vitro conditions, as well as the expression of all biosynthetic and regulatory genes of the DHN–melanin pathway in the three main Monilinia species upon exposure to light conditions (white, black, blue, red, and far-red wavelengths). On the other hand, we analyzed, for the first time, the metabolism related to ROS in M. fructicola, through the production of hydrogen peroxide (H2O2) and the expression of stress-related genes under different light conditions. In general, the results indicated a clear importance of black light on melanin production and expression in M. laxa and M. fructicola, but not in M. fructigena. Regarding ROS-related metabolism in M. fructicola, blue light highlighted by inhibiting the expression of many antioxidant genes. Overall, it represents a global description of the effect of light on the regulation of two important secondary mechanisms, essential for the adaptation of the fungus to the environment and its survival

Phenotypic plasticity of Monilinia spp. in response to light wavelengths: From in vitro development to virulence on nectarines

The development of brown rot in stone fruit caused by the necrotrophic fungus Monilinia spp. is influenced by many abiotic factors, such as temperature, humidity, and light. Specifically, filamentous fungi perceive light as a signal for ecophysiological and adaptive responses. We have explored how specific light wavelengths affect the in vitro development, the regulation of putative development genes and the virulence of the main species of Monilinia (M. laxa, M. fructicola and M. fructigena). After subjecting Monilinia spp. to different light wavelengths (white, black, blue, red, far-red) for 7 days, several differences in their phenotype were observed among light conditions, but also among species. These species of Monilinia exhibited a different phenotypic plasticity in response to light regarding pigmentation, growth, and specially conidiation of colonies. In this sense, we observed that the conidial production was higher in M. laxa than M. fructicola, while M. fructigena showed an inability to produce conidia under the tested conditions. Growth rate among species was significantly lower in M. fructicola under red light wavelength while among light conditions it was increased under far-red light wavelength for M. laxa and under black light for M. fructicola; in contrast, no statistical differences were observed for M. fructigena. Gene expression analysis of 13 genes involved in fungal development of Monilinia spp. revealed a significant difference among the three species of Monilinia, and especially depended on light wavelengths. Among them, a high expression of OPT1, RGS2, RGS3 and SPP1 genes was observed in M. laxa, and LTF1 and STE12 in M. fructicola under black light. In contrast, a high expression of REG1 and C6TF1 genes occurred in both M. fructicola and M. laxa subject to red and far-red light wavelength, respectively. When nectarines were artificially infected with M. laxa and M. fructicola subjected to black light, the virulence was clearly reduced, but not in M. fructigena. Overall, results presented herein demonstrate that light wavelengths are a key abiotic factor for the biology of Monilinia spp., specially modulating its capacity to form conidia, and thus, influencing its spreading and the onset of the disease on nectarines during postharvestinfo:eu-repo/semantics/acceptedVersio

Identification and Biosynthesis of DHN-melanin Related Pigments in the Pathogenic Fungi Monilinia laxa, M. fructicola, and M. fructigena

Monilinia is the causal agent of brown rot in stone fruit. The three main species that cause this disease are Monilinia laxa, M. fructicola, and M. fructigena, and their infection capacity is influenced by environmental factors (i.e., light, temperature, and humidity). To tolerate stressful environmental conditions, fungi can produce secondary metabolites. Particularly, melanin-like pigments can contribute to survival in unfavorable conditions. In many fungi, this pigment is due to the accumulation of 1,8-dihydroxynaphthalene melanin (DHN). In this study, we have identified for the first time the genes involved in the DHN pathway in the three main Monilinia spp. and we have proved their capacity to synthetize melanin-like pigments, both in synthetic medium and in nectarines at three stages of brown rot development. The expression of all the biosynthetic and regulatory genes of the DHN-melanin pathway has also been determined under both in vitro and in vivo conditions. Finally, we have analyzed the role of three genes involved in fungi survival and detoxification, and we have proved that there exists a close relationship between the synthesis of these pigments and the activation of the SSP1 gene. Overall, these results deeply describe the importance of DHN-melanin in the three main species of Monilinia: M. laxa, M. fructicola, and M. fructigena

Identification and Biosynthesis of DHN-melanin Related Pigments in the Pathogenic Fungi Monilinia laxa, M. fructicola, and M. fructigena

Monilinia is the causal agent of brown rot in stone fruit. The three main species that cause this disease are Monilinia laxa, M. fructicola, and M. fructigena, and their infection capacity is influenced by environmental factors (i.e., light, temperature, and humidity). To tolerate stressful environmental conditions, fungi can produce secondary metabolites. Particularly, melanin-like pigments can contribute to survival in unfavorable conditions. In many fungi, this pigment is due to the accumulation of 1,8-dihydroxynaphthalene melanin (DHN). In this study, we have identified for the first time the genes involved in the DHN pathway in the three main Monilinia spp. and we have proved their capacity to synthetize melanin-like pigments, both in synthetic medium and in nectarines at three stages of brown rot development. The expression of all the biosynthetic and regulatory genes of the DHN-melanin pathway has also been determined under both in vitro and in vivo conditions. Finally, we have analyzed the role of three genes involved in fungi survival and detoxification, and we have proved that there exists a close relationship between the synthesis of these pigments and the activation of the SSP1 gene. Overall, these results deeply describe the importance of DHN-melanin in the three main species of Monilinia: M. laxa, M. fructicola, and M. fructigena.info:eu-repo/semantics/publishedVersio