Labeling of Monilinia fructicola with GFP and Its Validation for Studies on Host-Pathogen Interactions in Stone and Pome Fruit

To compare in vivo the infection process of Monilinia fructicola on nectarines and apples using confocal microscopy it is necessary to transform a pathogenic strain with a construct expressing a fluorescent chromophore such as GFP. Thus, germinated conidia of the pathogen were transformed with Agrobacterium tumefaciens carrying the plasmid pPK2-hphgfp that allowed the expression of a fluorescent Hph-GFP chimera. The transformants were selected according to their resistance to hygromycin B, provided by the constitutive expression of the hph-gfp gene driven by the glyceraldehyde 3P dehydrogenase promoter of Aspergillus nidulans. The presence of T-DNA construct in the genomic DNA was confirmed by PCR using a range of specific primers. Subsequent PCR-mediated analyses proved integration of the transgene at a different genomic location in each transformant and the existence of structural reorganizations at these insertion points. The expression of Hph-GFP in three independent M. fructicola transformants was monitored by immunodetection and epifluorescence and confocal microscopy. The Atd9-M. fructicola transformant displayed no morphological defects and showed growth and pathogenic characteristics similar to the wild type. Microscopy analysis of the Atd9 transformant evidenced that nectarine infection by M. fructicola was at least three times faster than on applesinfo:eu-repo/semantics/publishedVersio

Exploring sources of resistance to brown rot in an interspecific almond × peach population

Background: Monilinia spp. are responsible for brown rot, one of the most significant stone fruit diseases. Planting resistant cultivars seems a promising alternative, although most commercial cultivars are susceptible to brown rot. The aim of this study was to explore resistance to Monilinia fructicola over two seasons in a backcross one interspecific population between almond 'Texas' and peach 'Earlygold' (named T1E). - Results: 'Texas' almond was resistant to brown rot inoculation, whereas peach was highly susceptible. Phenotypic data from the T1E population indicated wide differences in response to M. fructicola. Additionally, several non-wounded individuals exhibited resistance to brown rot. Quantitative trait loci (QTLs) were identified in several linkage groups, but only two proximal QTLs in G4 were detected over both seasons and accounted for 11.3-16.2% of the phenotypic variation. - Conclusion: analysis of the progeny allowed the identification of resistant genotypes that could serve as a source of resistance in peach breeding programs. The finding of loci associated with brown rot resistance would shed light on implementing a strategy based on marker-assisted selection (MAS) for introgression of this trait into elite peach materials. New peach cultivars resistant to brown rot may contribute to the implementation of more sustainable crop protection strategies

Pectin methyl esterases and rhamnogalacturonan hydrolases: weapons for successful Monilinia laxa infection in stone fruit?

The secretion of cell wall‐degrading enzymes is one of the mechanisms used by necrotrophic fungi to colonize host tissues. However, information about virulence factors of Monilinia spp., the causal agents of brown rot in stone fruit, is scarce. Plant cell walls have three main components that are broken down by fungal enzymes: cellulose, hemicellulose and pectin. In order to identify Monilinia laxa candidate proteins involved in pectin hydrolysis, two in vitro approaches were conducted: (i) phenotypic and ecophysiological characterization of growth of the pathogen at different pHs, in glucose‐ and pectin‐containing solid media for 7 days' incubation; and (ii) expression analysis of genes encoding M. laxa pectin methyl esterases (MlPMEs) and rhamnogalacturonan hydrolases (MlRG‐HYDs) after incubation for 0.5, 2, 6, 24 and 48 h in glucose‐ and pectin‐containing liquid media. Phenotypic tests showed the role of carbon source on M. laxa growth rate and aggressiveness, and indicated that pectinases were greatly affected by pH. Gene expression analyses uncovered differences among members of each family of pectinases and between the two families, defining sets of genes expressed at earlier (0.5–6 h) and later (48 h) phases. Notably, the up‐ or down‐regulation of these target genes was carbon source‐dependent. Finally, an in vivo study confirmed the synergistic and complementary role that these genes play in the M. laxa–stone fruit pathosystem. Based on these results, it is hypothesized that MlPME2, MlRG‐HYD1 and MlRG‐HYD2 may be potential virulence factors of M. laxa in the process from infection to colonization.info:eu-repo/semantics/acceptedVersio

Noves aproximacions en els estudis d'interacció de fruita de pinyol i Monilinia spp. per al control sostenible de la podridura marró

El control de plagues, malalties i fisiopaties en fruiters és cada cop més complex, i la podridura marró no n’és una excepció. Aquesta malaltia, causada per Monilinia spp., afecta la fruita de pinyol de la nostra zona, ocasionant greus pèrdues al camp i en postcollita. Actualment, la principal estratègia per al control d’aquesta malaltia es basa en programes d’aplicació de productes fungicides de síntesi en el marc d’una producció integrada. No obstant això, la conscienciació d’una bona part de la societat sobre la seguretat dels aliments i la protecció del medi ambient fa necessari cercar noves aproximacions multidisciplinàries, com les que es proposen en aquesta tesi, tenint en compte les dues parts implicades: l’hoste (fruita de pinyol) i el patogen (Monilinia spp.), les quals ajuden a incrementar el coneixement de la malaltia i per tant, a millorar l’eficàcia i l’efectivitat dels tractaments. Globalment, el coneixement generat en aquesta tesi permetrà seguir avançant en la cerca de noves aproximacions com, per exemple, el desenvolupament de varietats menys susceptibles a la podridura marró; també saber com Monilinia spp. pot modular mecanismes de defensa del fruit; i, per últim, definir factors de virulència claus per a la infecció. Per tant, aquests estudis aporten noves perspectives en el disseny de tractaments de control de la podridura marró, així com nous recursos per a millorar l’eficàcia i l’efectivitat dels ja existents.El control de plagas, enfermedades y fisiopatías en frutales es cada vez más complejo, y la podredumbre parda no es una excepción. Esta enfermedad, causada por Monilinia spp., afecta a la fruta de hueso de nuestra zona, ocasionando graves pérdidas en campo y poscosecha. Actualmente, la principal estrategia para su control se basa en la aplicación de productos fungicidas de síntesis en el marco legislativo de una producción integrada. Sin embargo, la concienciación de la sociedad por la seguridad de los alimentos y la protección del medio ambiente requiere nuevas aproximaciones multidisciplinares, como las que se proponen en la presente tesis, que consideren ambas partes implicadas: el huésped (fruta de hueso) y el patógeno (Monilinia spp.) con el propósito de aumentar el conocimiento de la enfermedad, y por tanto, mejorar la eficacia y efectividad de los tratamientos. La integración del conocimiento generado en esta tesis permitirá seguir avanzando en la búsqueda de nuevas aproximaciones como, por ejemplo, el desarrollo de variedades menos susceptibles a la podredumbre parda; saber cómo Monilinia spp. puede modular mecanismos de defensa del fruto; y, por último, definir factores de virulencia claves para la infección. Por lo tanto, estos estudios aportan nuevas perspectivas en el diseño de tratamientos de control de la podredumbre marrón, así como nuevos recursos para mejorar la eficacia y la efectividad de los ya existentes.The control of fruit pests and diseases is becoming increasingly complex, and brown rot is not an exception. This disease, caused by the ascomycete Monilinia spp., is an important stone fruit disease in our area, responsible for losses in the entire fruit production chain. The current strategy to control brown rot is based on cultural practices and the use of fungicide spray programmes in the field, followed in some cases by a postharvest treatment. However, because of restrictions on the use of fungicides and consumers’ concerns about health risks and environmental contamination, further research is needed. In this context, new multidisciplinary approaches such as the ones that are presented in this thesis, that consider both: the host (stone fruit) and the pathogen (Monilinia spp.), would allow for a better understanding of the disease and hence, to more specific design and effective crop protection strategies. The knowledge generated in this thesis provide a framework for developing more rational control alternatives to synthetic fungicides, such as the development of resistant varieties to brown rot. Besides, knowing how Monilinia spp. can modulate fruit defence mechanisms of the fruit and, unravelling virulence factors that are key to the infection will allow for a better understanding of the disease and hence, to more specific and effective crop protection strategies

Noves aproximacions en els estudis d'interacció de fruita de pinyol i Monilinia spp. per al control sostenible de la podridura marró

El control de plagues, malalties i fisiopaties en fruiters és cada cop més complex, i la podridura marró no n’és una excepció. Aquesta malaltia, causada per Monilinia spp., afecta la fruita de pinyol de la nostra zona, ocasionant greus pèrdues al camp i en postcollita. Actualment, la principal estratègia per al control d’aquesta malaltia es basa en programes d’aplicació de productes fungicides de síntesi en el marc d’una producció integrada. No obstant això, la conscienciació d’una bona part de la societat sobre la seguretat dels aliments i la protecció del medi ambient fa necessari cercar noves aproximacions multidisciplinàries, com les que es proposen en aquesta tesi, tenint en compte les dues parts implicades: l’hoste (fruita de pinyol) i el patogen (Monilinia spp.), les quals ajuden a incrementar el coneixement de la malaltia i per tant, a millorar l’eficàcia i l’efectivitat dels tractaments. Globalment, el coneixement generat en aquesta tesi permetrà seguir avançant en la cerca de noves aproximacions com, per exemple, el desenvolupament de varietats menys susceptibles a la podridura marró; també saber com Monilinia spp. pot modular mecanismes de defensa del fruit; i, per últim, definir factors de virulència claus per a la infecció. Per tant, aquests estudis aporten noves perspectives en el disseny de tractaments de control de la podridura marró, així com nous recursos per a millorar l’eficàcia i l’efectivitat dels ja existents.El control de plagas, enfermedades y fisiopatías en frutales es cada vez más complejo, y la podredumbre parda no es una excepción. Esta enfermedad, causada por Monilinia spp., afecta a la fruta de hueso de nuestra zona, ocasionando graves pérdidas en campo y poscosecha. Actualmente, la principal estrategia para su control se basa en la aplicación de productos fungicidas de síntesis en el marco legislativo de una producción integrada. Sin embargo, la concienciación de la sociedad por la seguridad de los alimentos y la protección del medio ambiente requiere nuevas aproximaciones multidisciplinares, como las que se proponen en la presente tesis, que consideren ambas partes implicadas: el huésped (fruta de hueso) y el patógeno (Monilinia spp.) con el propósito de aumentar el conocimiento de la enfermedad, y por tanto, mejorar la eficacia y efectividad de los tratamientos. La integración del conocimiento generado en esta tesis permitirá seguir avanzando en la búsqueda de nuevas aproximaciones como, por ejemplo, el desarrollo de variedades menos susceptibles a la podredumbre parda; saber cómo Monilinia spp. puede modular mecanismos de defensa del fruto; y, por último, definir factores de virulencia claves para la infección. Por lo tanto, estos estudios aportan nuevas perspectivas en el diseño de tratamientos de control de la podredumbre marrón, así como nuevos recursos para mejorar la eficacia y la efectividad de los ya existentes.The control of fruit pests and diseases is becoming increasingly complex, and brown rot is not an exception. This disease, caused by the ascomycete Monilinia spp., is an important stone fruit disease in our area, responsible for losses in the entire fruit production chain. The current strategy to control brown rot is based on cultural practices and the use of fungicide spray programmes in the field, followed in some cases by a postharvest treatment. However, because of restrictions on the use of fungicides and consumers’ concerns about health risks and environmental contamination, further research is needed. In this context, new multidisciplinary approaches such as the ones that are presented in this thesis, that consider both: the host (stone fruit) and the pathogen (Monilinia spp.), would allow for a better understanding of the disease and hence, to more specific design and effective crop protection strategies. The knowledge generated in this thesis provide a framework for developing more rational control alternatives to synthetic fungicides, such as the development of resistant varieties to brown rot. Besides, knowing how Monilinia spp. can modulate fruit defence mechanisms of the fruit and, unravelling virulence factors that are key to the infection will allow for a better understanding of the disease and hence, to more specific and effective crop protection strategies

Scrutinising the relationship between major physiological and compositional changes during ‘Merrill O’Henry’ peach growth with brown rot susceptibility

In the present work, the major physiological and compositional changes occurring during 'Merrill O'Henry' peach growth and its relationship with susceptibility to three strains of Monilinia spp. at 49, 77, 126 and 160 days after full bloom were explored. Results of disease incidence indicated wide differences among phenological stages, being 49 and 126 days after full bloom the moment when peaches showed significantly lower susceptibility to brown rot (40 and 23% of rotten fruit, respectively, for strain ML8L). Variation in brown rot susceptibility among different growth stages was also strain-dependent. Lower fruit susceptibility to ML8L at 49 and 126 was accompanied by noticeable changes in the fruit ethylene and respiration patterns, and also in sugars and organic acids content. By employing a partial least squares regression model, a strong negative relationship between citric acid, and a positive association of ethylene with peach susceptibility to Monilinia spp. at diverse phenological stages were observed. The results obtained herein highlight that the content of certain compounds such as citrate, malate and sucrose; the respiratory activity and the fruit ethylene production may mediate in a coordinated manner the fruit resistance to Monilinia spp. at different phenological stages of peach fruit.info:eu-repo/semantics/acceptedVersio

A walk-through method for identifying brown rot resistance in stone fruit: methodology development, validation, and application on an interspecific almond × peach population

Resumen del trabajo presentado al Vth International Symposium on Postharvest Pathology, celebrado en Liège (Bélgica) del 19 al 24 de mayo de 2019.Authors are grateful to Spanish Government for their financial support by national projects AGL2014-55287-C02-02 and RTA2015-00050-00-00 from Ministry of Economy, Industry and Competitiveness (MINECO) and to the Catalan Government (Generalitat de Catalunya) for the PhD grant 2016FI_B_00442 (Baró-Montel, N.) and for the funding received from CERCA Programme / Generalitat de Catalunya. Thanks are also given to Severo Ochoa Program for Centres of Excellence in R&D 2016-2019 (SEV-2015-0533)

Double-sided battle: The role of ethylene during Monilinia spp. infection in peach at different phenological stages

Controversy exists on whether ethylene is involved in determining fruit resistance or susceptibility against biotic stress. In this work, the hypothesis that ethylene biosynthesis in peaches at different phenological stages may be modulated by Monilinia spp. was tested. To achieve this, at 49 and 126 d after full bloom (DAFB), ethylene biosynthesis of healthy and infected ‘Merryl O'Henry’ peaches with three strains of Monilinia spp. (M. fructicola (CPMC6) and M. laxa (CPML11 and ML8L) that differ in terms of aggressiveness) was analysed at the biochemical and molecular level along the course of infection in fruit stored at 20 °C. At 49 DAFB, results evidenced that infected fruit showed inhibition of ethylene production in comparison with non-inoculated fruit, suggesting that the three Monilinia strains were somehow suppressing ethylene biosynthesis to modify fruit defences to successfully infect the host. On the contrary, at 126 DAFB ethylene production increased concomitantly with brown rot spread, and values for non-inoculated fruit were almost undetectable throughout storage at 20 °C. The expression of several target genes involved in the ethylene biosynthetic pathway confirmed that they were differentially expressed upon Monilinia infection, pointing to a strain-dependent regulation. Notably, Prunus persica 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS) (PpACS) family was the most over-expressed over time, demonstrating a positive ethylene regulation, especially at 126 DAFB. At this phenological stage it was demonstrated the ability of Monilinia spp. to alter ethylene biosynthesis through PpACS1 and benefit from the consequences of an ethylene burst likely on cell wall softening. Overall, our results put forward that infection not only among different strains but also at each stage is achieved by different mechanisms, with ethylene being a key factor in determining peach resistance or susceptibility to brown rot.info:eu-repo/semantics/acceptedVersio