Resistencia frente a la infección por Penicillium digitatum durante la postcosecha de los frutos cítricos. Bases moleculares y metabolismo de fenilpropanoides.

RESUMEN Penicillium digitatum es el agente causal de la podredumbre verde de los frutos cítricos y constituye uno de los patógenos más importantes durante la postcosecha de los mismos, llegando a causar hasta el 80 % de las podredumbres. Aunque el control de este patógeno se realiza en la actualidad con fungicidas químicos, existe una tendencia al abandono de los mismos y al empleo de estrategias alternativas de control. La inducción de resistencia natural en los frutos constituye una de estas alternativas. En el presente trabajo se pretende profundizar en las bases biológicas y moleculares de la inducción de resistencia en los frutos cítricos frente a un ataque por P. digitatum. Se han evaluado distintos métodos de inducción de resistencia frente a P. digitatum con el fin de optimizar y seleccionar uno de ellos con el que abordar los estudios posteriores. Se compararon diversos tratamientos químicos (metil jasmonato, ácido β aminobutírico y Brotomax®) y biológicos (hongos no patógenos de cítricos y paredes celulares de P. digitatum), así como un tratamiento de infección previa con P. digitatum y posterior inactivación térmica del patógeno. Se seleccionó este último sistema atendiendo a los buenos niveles de resistencia alcanzados (la incidencia se redujo más de un 70 % y la severidad más de un 85 %) y a la reproducibilidad del mismo. El tratamiento seleccionado se empleó para estudiar los cambios globales de expresión génica durante la inducción de resistencia. Estos cambios se analizaron en el flavedo y el albedo de frutos cítricos empleando dos micromatrices de cDNA desarrolladas por el Consorcio de Genómica Funcional de Cítricos, denominadas 7k y 12k, que contienen aproximadamente 7.000 y 12.000 genes respectivamente. Las mayores inducciones se detectaron en genes que codifican enzimas del metabolismo de fenilpropanoides, especialmente metiltransferasas (OMTs), de la síntesis de etileno, entre los que se encuentran varios genes ACO (ácido 1-metilciclopropano 1-carboxílico oxidasa), proteínas relacionadas con la patogénesis (proteínas PR) y oxidorreductasas. También se observó una importante reducción de la expresión de genes regulados por bajas temperaturas, de genes inducibles por la luz y de otros de función desconocida. El análisis de anotación funcional por ontologías génicas mostró que los procesos relacionados con el metabolismo de aminoácidos y de fenilpropanoides se indujeron en los frutos sometidos al tratamiento de inducción de resistencia respecto a los frutos no tratados, siendo estos cambios mayores en el tejido interno. Una vez finalizado el tratamiento de inducción de resistencia tiene lugar un aumento de la producción de etileno, que es comparable al que se observa en frutos infectados por P. digitatum. Sin embargo, en los frutos en los que se induce resistencia no hay desarrollo del hongo, por lo que todo el etileno procede del fruto. Mediante análisis Northern se ha comprobado que este aumento de los niveles de etileno coincide con una elevada inducción de la expresión del gen CsACO, al igual que ocurre en frutos infectados. Es de reseñar que las mayores inducciones tienen lugar en el albedo. Para profundizar en el estudio de la implicación de la ruta de fenilpropanoides se procedió a analizar la expresión de genes que codifican las enzimas PAL, 4CL, F3H, IRL1, CADs, SAD y OMTs mediante hibridación Northern. Se observó que la expresión de la mayor parte de estos genes aumentaba en respuesta al tratamiento inductor, con un patrón similar en ambos tejidos. Aunque en general los niveles de expresión fueron superiores en el flavedo, las mayores inducciones se detectaron en el albedo. Además, se realizó un estudio de la expresión de estos genes en respuesta a la infección por P. digitatum, observándose que los cambios fueron, en general, similares a los inducidos por el tratamiento de inducción de resistencia. El estudio de esta ruta se completó con la determinación del perfil metabólico de compuestos secundarios en los frutos cítricos mediante HPLC. En los frutos no tratados se observó en general una mayor abundancia y concentración de compuestos fenólicos en el flavedo respecto al albedo. Sólo la concentración de algunas flavanonas fue mayor en el albedo. La escoparona, 6,7-dihidroxicumarina, fue el compuesto con mayor inducción en respuesta al tratamiento inductor. Sin embargo, el mayor número de cambios como consecuencia de la infección y posterior inactivación térmica se observó en los derivados del ácido benzoico. Al igual que en respuesta al tratamiento inductor, durante la infección por P. digitatum se observó una modificación en la concentración de derivados del ácido benzoico. También se ha analizado la posible actividad antifúngica in vitro e in vivo de diversas cumarinas, flavanonas y alcaloides sobre diferentes hongos patógenos. Las cumarinas escoparona y umbeliferona y la flavanona hesperidina fueron los compuestos con mayor actividad antifúngica in vitro, aunque ninguno de ellos inhibió por completo el crecimiento de los hongos patógenos de cítricos. Sin embargo, sólo las dos cumarinas mostraron un efecto in vivo retrasando el desarrollo de P. digitatum en frutos inoculados con este patógeno. La adición al medio de cultivo de algunos alcaloides presentes en los cítricos redujo significativamente el crecimiento de P. digitatum, siendo la cafeina y el ácido 1, 2, 3, 4-tetrahidroharmane 3-carboxílico (THHCA) los compuestos más efectivos. Este trabajo constituye la primera aproximación en el que se aborda conjuntamente un análisis transcriptómico y metabolómico de procesos relevantes en patología postcosecha. Los resultados obtenidos ponen de manifiesto la importancia que desempeñan tanto el metabolismo secundario, especialmente de fenilpropanoides, como el etileno en el proceso de resistencia inducida en frutos cítricos frente a la infección por P. digitatum. __________________________________________________________________________________________________Penicillium digitatum is the causal agent of green mould and constitutes the major postharvest pathogen of citrus fruit, accounting for up to 80 % of losses due to fungal decay. Although control of this pathogen is achieved by chemical fungicides, there is a trend to adopt new and safer control alternatives. Induction of natural resistance in fruit constitutes one of these alternatives. The aim of this work was to gain an insight into the biological and molecular bases of the induction of resistance in citrus fruit against P. digitatum. Different methods to induce resistance against P. digitatum have been evaluated in order to optimize and to select one of them for further studies. Various chemical (methyl jasmonate, β-aminobutyric acid and Brotomax®), biological (non pathogenic fungi of citrus and cellular walls of P. digitatum) treatments, as well as the possible resistance induced by a previous infection with P. digitatum followed by its inactivation by a heat treatment were compared. The latter system was selected because of the high resistance achieved (the incidence was reduced more than 70 % and the severity more than 85 %) and its reproducibility. Global changes in gene expression induced by the selected treatment were analyzed in the flavedo and albedo of citrus fruit. To that end, two cDNA microarrays developed by the Citrus Functional Genomics Project, named 7k and 12k, which contain about 7.000 and 12.000 genes, respectively, were used. The highest inductions were detected in genes that code for enzymes involved in phenylpropanoid metabolism, mainly methyltransferases (OMTs), ethylene biosynthesis, among which there are several ACO genes (1-aminocyclopropane 1-carboxylic acid oxidase), pathogenesis-related proteins (PR) and oxidoreductases. An important reduction in the expression of genes regulated by either cold or light, together with others of unknown function, was also observed. Gene ontology comparisons showed that processes related to amino acids and phenylpropanoid metabolism were upregulated in treated fruits, being these changes more marked in the internal tissue. An increase in ethylene production was observed after the heat treatment, coincident with the resistance acquisition, reaching a concentration similar to that observed in fruits infected by P. digitatum. Nevertheless, in the treated fruits the fungus does not progress, so the rise in ethylene must be due to the fruit. Northern blot analysis showed that this increase in ethylene paralleled a high induction on CsACO expression, as it occurs in infected fruits, and that the highest induction takes place into the albedo. In order to get a deeper insight into the role of the phenylpropanoid metabolism in the induction of resistance, the expression of genes encoding PAL, 4CL, F3H, IRL1, CADs, SAD and OMTs was analyzed by Northern blot. The expression of most of these genes increased in response to the induction treatment, showing a similar pattern in both tissues. Although the overall expression levels were higher in the flavedo, the highest inductions were detected in the albedo. In addition, changes in the expression of these genes in response to the infection by P. digitatum were similar to those originated by the induction of resistance treatment. The putative involvement of this pathway was further studied by examining changes in the metabolic profile by means of HPLC. In the treated fruits a greater abundance and a higher concentration of phenolic compounds in the flavedo with respect to the albedo were observed. Flavanones were the only phenolics whose concentration was higher in the internal tissue. Although the coumarin scoparone was the phenolic compound showing the highest increase in response to the treatment, it has to be pointed out that, among the different phenylpropanoids, benzoic acid derivatives were the most affected. Similar changes were detected during the infection of fruits by P. digitatum. The possible antifungal activity of diverse coumarins, flavanones and alkaloids was tested both in vitro and in vivo against different pathogenic fungi. The coumarins scoparone and umbelliferone and the flavanone hesperidin showed the highest in vitro antifungal activity, although none of them inhibited completely the growth of the citrus fungal pathogens analyzed. However, only the coumarins had an effect in vivo delaying the development of P. digitatum in inoculated citrus fruit. On the other hand, addition of some alkaloids detected in citrus tissues to the culture medium significantly reduced the growth of P. digitatum, being caffeine and 1, 2, 3, 4-tetrahydroharmane 3-carboxylic acid (THHCA) the most effective. This is the first work undertaking both a transcriptomic and a metabolomic approach to study changes associated with relevant processes in postharvest pathology. The global results indicate that both the secondary metabolism, mainly phenylpropanoids, and ethylene play important roles in the induction of resistance in citrus fruit against P. digitatum infection

Identification and functional analysis of Penicillium digitatum genes putatively involved in virulence towards citrus fruit

The fungus Penicillium digitatum, the causal agent of green mould rot, is the most destructive post-harvest pathogen of citrus fruit in Mediterranean regions. In order to identify P.digitatum genes up-regulated during the infection of oranges that may constitute putative virulence factors, we followed a polymerase chain reaction (PCR)-based suppression subtractive hybridization and cDNA macroarray hybridization approach. The origin of expressed sequence tags (ESTs) was determined by comparison against the available genome sequences of both organisms. Genes coding for fungal proteases and plant cell wall-degrading enzymes represent the largest categories in the subtracted cDNA library. Northern blot analysis of a selection of P.digitatum genes, including those coding for proteases, cell wall-related enzymes, redox homoeostasis and detoxification processes, confirmed their up-regulation at varying time points during the infection process. Agrobacterium tumefaciens-mediated transformation was used to generate knockout mutants for two genes encoding a pectin lyase (Pnl1) and a naphthalene dioxygenase (Ndo1). Two independent P.digitatum Δndo1 mutants were as virulent as the wild-type. However, the two Δpnl1 mutants analysed were less virulent than the parental strain or an ectopic transformant. Together, these results provide a significant advance in our understanding of the putative determinants of the virulence mechanisms of P.digitatum. Molecular Plant PathologyLGC research is funded in part by the Spanish Ministry of Economy and Innovation (AGL-2008-04828-C03-02 and AGL2011-30519-C03-01) and the Generalitat Valenciana (PROMETEO/2010/010, PROMETEOII/2014/027 and ACOMP/2011/250). MLP was supported by a FPI scholarship (BES-2006-12983). ARB acknowledges the support of the JAE-Doc program from CSIC cofounded by FSE.Peer Reviewe

Molecular aspects in pathogen-fruit interactions: Virulence and resistance

Fruit losses during postharvest storage and handling due to pathogen infections are one of the major problems in the global food chain supply. The application of chemical fungicides to control diseases is currently limited by legislation in some countries and also raises concerns about food and environmental safety. Exploring molecular aspects of pathogen-fruit interactions therefore has biological and economic significance as a means to help develop rational alternatives for disease control. In this review we present the current knowledge of molecular aspects in pathogen-fruit interactions, addressing the following topics: the application of new “omics” technologies for studying these interactions; the molecular mechanisms of fungal pathogen attack; the regulation of virulence by exogenous factors; and, finally, fruit defense mechanisms.Work in Tian’s lab has been funded by the National Natural Science Foundation of China (31530057; 31371863). Work in the LGC lab has been funded by the Spanish Ministry of Economy and Competitiveness (AGL2011-30519-C03-01 and AGL2014-55802-R) and the Generalitat Valenciana (PrometeoII/2014/027). Work in the IRTA lab has been funded by the Spanish Ministry of Economy and Competitiveness by three national projects AGL2008-04828-C01/AGR, AGL2011-30519-C03/AGR and AGL2014-55287-C02.Peer reviewe

Citrus phenylpropanoids and defence against pathogens. Part I: Metabolic profiling in elicited fruits

Penicillium spp. are among the major postharvest pathogens of citrus fruit. Induction of natural resistance in fruits constitutes one of the alternatives to chemical fungicides. Here, we investigated the involvement of the phenylpropanoid pathway in the induction of resistance in Navelate oranges by examining changes in the metabolic profile of upon eliciting citrus fruits. By using both HPLC-PDA-FD and HPLC-PDA-QTOF-MS allowed the identification of several compounds that seem to be relevant for induced resistance. In elicited fruits, a greater diversity of phenolic compounds was observed in the flavedo (outer coloured part of the peel) when compared to the albedo (inner white part). Moreover, only small changes were detected in the most abundant citrus flavonoids. The coumarin scoparone was among the compounds with the highest induction upon elicitation. Two other highly induced compounds were identified as citrusnin A and drupanin aldehyde. All three compounds are known to exert antimicrobial activity. Our results suggest that phenylpropanoids and their derivatives play an important role in the induction of resistance in citrus fruit.This work was supported by Research Grants AGL2008-04828-C03-02, AGL2009-11969 and CONSOLIDER FUNC-FOOD from the Spanish Ministry of Science and Technology, and PROMETEO/2010/010 from the Generalitat Valenciana.Peer Reviewe

Inhibiting ethylene perception with 1-methylcyclopropene triggers molecular responses aimed to cope with cell toxicity and increased respiration in citrus fruits

The ethylene perception inhibitor 1-methylcyclopropene (1-MCP) has been critical in understanding the hormone's mode of action. However, 1-MCP may trigger other processes that could vary the interpretation of results related until now to ethylene, which we aim to understand by using transcriptomic analysis. Transcriptomic changes in ethylene and 1-MCP-treated ‘Navelate’ (Citrus sinensis L. Osbeck) oranges were studied in parallel with changes in ethylene production, respiration and peel damage. The effects of compounds modifying the levels of the ethylene co-product cyanide and nitric oxide (NO) on fruit physiology were also studied. Results suggested that: 1) The ethylene treatment caused sub-lethal stress since it induced stress-related responses and reduced peel damage; 2) 1-MCP induced ethylenedependent and ethylene-independent responsive networks; 3) 1-MCP triggered ethylene overproduction, stress-related responses and metabolic shifts aimed to cope with cell toxicity, which mostly affected to the inner part of the peel (albedo); 4) 1-MCP increased respiration and drove metabolism reconfiguration for favoring energy conservation but up-regulated genes related to lipid and protein degradation and triggered the over-expression of genes associated with the plasma membrane cellular component; 5) Xenobiotics and/or reactive oxygen species (ROS) might act as signals for defense responses in the ethylene-treated fruit, while their uncontrolled generation would induce processes mimicking cell death and damage in 1-MCP-treated fruit; 6) ROS, the ethylene co-product cyanide and NO may converge in the toxic effects of 1-MCP.This work was supported by the Spanish Ministry of Science and Technology (Research Grants AGL2002-1727 and AGL2009-11969 and by the Generalitat Valenciana, Spain (Grant PROMETEOII/2014/027). Dr. B. E. was the recipient of a fellowship of the Spanish Ministry of Science and Technology. Dr. A.R.B. is grateful to CSIC and the European Social Fund for her postdoctoral JAE-Doc contract.Peer reviewe

Unravelling molecular responses to moderate dehydration in harvested fruit of sweet orange (Citrus sinensis L. Osbeck) using a fruit-specific ABA-deficient mutant

Water stress affects many agronomic traits that may be regulated by the phytohormone abscisic acid (ABA). Within these traits, loss of fruit quality becomes important in many citrus cultivars that develop peel damage in response to dehydration. To study peel dehydration transcriptional responsiveness in harvested citrus fruit and the putative role of ABA in this process, this study performed a comparative large-scale transcriptional analysis of water-stressed fruits of the wild-type Navelate orange (Citrus sinesis L. Osbeck) and its spontaneous ABA-deficient mutant Pinalate, which is more prone to dehydration and to developing peel damage. Major changes in gene expression occurring in the wild-type line were impaired in the mutant fruit. Gene ontology analysis revealed the ability of Navelate fruits to induce the response to water deprivation and di-, tri-valent inorganic cation transport biological processes, as well as repression of the carbohydrate biosynthesis process in the mutant. Exogenous ABA triggered relevant transcriptional changes and repressed the protein ubiquitination process, although it could not fully rescue the physiological behaviour of the mutant. Overall, the results indicated that dehydration responsiveness requires ABA-dependent and-independent signals, and highlight that the ability of citrus fruits to trigger molecular responses against dehydration is an important factor in reducing their susceptibility to developing peel damage.This work was supported by the Spanish Ministry of Science and Technology (Research Grants AGL2006-09496, AGL2009-11969, and AGL2009-11558) and by the Generalitat Valenciana (PROMETEO/2010/010). PR was the recipient of a fellowship from the Spanish Ministry of Science and Technology.Peer Reviewe

Differential contribution of the two major polygalacturonases from Penicillium digitatum to virulence towards citrus fruit

The fungus Penicillium digitatum is the causal agent of the citrus green mould, the major postharvest diseases of citrus fruit. Lesions on the surface of infected fruits first appear as soft areas around the inoculation site, due to maceration of fruit. The macerating activity has been associated with pectinases secreted by the fungus during infection. In order to evaluate the contribution to virulence and macerating activity of the two major polygalacturonases (PGs) secreted by P. digitatum, we have obtained and characterized mutants lacking either pg1 or pg2, the genes encoding PG1 and PG2, respectively. Disease incidence of deletants in either gene was not different from that of the parental strain or ectopic transformants. However, disease progressed more slowly in deletants, especially in those lacking the pg2 gene. The lesions originated by the Δpg2 deletants were not as soft and the pH was not as acid as those originated by either the wild type strain or the ectopic transformants. Total PG activity in the macerated tissue was also lower in fruits infected with the Δpg2 deletants. Interestingly, the macerated tissue of oranges infected with Δpg2 deletants showed around 50% reduction in galacturonic acid content with respect to lesions caused by any other strain.The technical assistance of Ana Izquierdo is gratefully acknowledged. LG-C's research is funded in part by the Spanish Ministry of Economy, Industry and Competitiveness (AGL2011-30519-C03-01 and AGL2014-55802-R) and the Generalitat Valenciana (PROMETEOII/2014/027). ML-P was supported by a “Formación de Personal Investigador” scholarship (BES-2006-12983). Authors want to thank the technical assistance of S. Dashevskaya and the financial support by AGL2011-30519-CO3-03 from the “Ministerio de Economía y Competitividad” (MINECO, Spain), and the CERCA Programme/Generalitat de Catalunya

Self-management interventions for adults living with obesity to improve patient-relevant outcomes : An evidence map

To conduct an evidence map on self-management interventions and patient-relevant outcomes for adults living with overweight/obesity. Following Arksey and O'Malley methodology, we searched in five electronical databases including randomized controlled trials (RCTs) on SMIs for overweight/obesity. We used the terms "self-management", "adult" and "obesity" for content. Two independent reviewers assessed eligible references; one reviewer extracted data, a second checked accuracy. We identified 497 RCTs (58% US, 20% Europe) including 99,741 (median 112, range 11-5145) adults living with overweight/obesity. Most research evaluated clinical outcomes (617, 55%) and behaviors adherence (255, 23%). Empowerment skills, quality of life and satisfaction were less targeted (8%, 7%, 0.2%, respectively). The most frequent techniques included sharing information (858, 99%), goal setting (619, 72%) and self-monitoring training (614, 71%), provided face-to-face (386, 45%) or in combination with remote techniques (256, 30%). Emotional management, social support and shared-decision were less frequent (18%, 26%, 4%). Socio-economic status, minorities or health literacy were seldom reported. There is a need of widening the scope of research by focusing on outcomes important to patients, assessing emotional/social/share-decision support, exploring remote techniques and including vulnerable populations

Using a Taxonomy to Systematically Identify and Describe Self-Management Interventions Components in Randomized Trials for COPD

Self-management interventions (SMIs) may improve outcomes in Chronic Obstructive Pulmonary Disease (COPD). However, accurate comparisons of their relative effectiveness are challenging, partly due to a lack of clarity and detail regarding the intervention content being evaluated. This study systematically describes intervention components and characteristics in randomized controlled trials (RCTs) related to COPD self-management using the COMPAR-EU taxonomy as a framework, identifying components that are insufficiently incorporated into the design of the intervention or insufficiently reported. Overall, 235 RCTs published between 2010 and 2018, from a systematic review were coded using the taxonomy, which includes 132 components across four domains: intervention characteristics, expected patient (or caregiver) self-management behaviours, patient relevant outcomes, and target population characteristics. Risk of bias was also assessed. Interventions mainly focused on physical activity (67.4%), and condition-specific behaviours like breathing exercise (63.5%), self-monitoring (50.8%), and medication use (33.9%). Support techniques like education and skills-training, self-monitoring, and goal setting (over 35% of the RCTs) were mostly used for this. Emotional-based techniques, problem-solving, and shared decision-making were less frequently reported (less than 15% of the studies). Numerous SMIs components were insufficiently incorporated into the design of COPD SMIs or insufficiently reported. Characteristics like mode of delivery, intensity, location, and providers involved were often not described. Only 8% of the interventions were tailored to the target population's characteristics. Outcomes that are considered important by patients were hardly taken into account. There is still a lot to improve in both the design and description of SMIs for COPD. Using a framework such as the COMPAR-EU SMI taxonomy may contribute to better reporting and to better informing of replication efforts. In addition, prospective use of the taxonomy for developing and reporting intervention content would further aid in building a cumulative science of effective SMIs in COPD