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Introduction: Brown rot is the most important fungal disease affecting stone fruit and it is mainly caused by Monilinia fructicola, M. laxa and M. fructigena. Monilinia spp. are necrotrophic plant pathogens with the ability to induce plant cell death by the secretion of different phytotoxic molecules, including proteins or metabolites that are collectively referred to as necrotrophic effectors (NEs). Methods: We exploited the genomes of M. fructicola, M. laxa and M. fructigena to identify their common group of secreted effector proteins and tested the ability of a selected set of effectors to induce cell death in Nicotiana benthamiana, Solanum lycopersicum and Prunus spp. leaves. Results: Fourteen candidate effector genes of M. fructicola, which displayed high expression during infection, were transiently expressed in plants by agroinfiltration using a modified Tobacco Rattle Virus (TRV)-based expression system. Some, but not all, effectors triggered leaf discoloration or cell death in N. benthamiana and S. lycopersicum, which are non-hosts for Monilinia and in Prunus spp., which are the natural hosts. The effector MFRU_030g00190 induced cell death in almost all Prunus genotypes tested, but not in the Solanaceous plants, while MFRU_014g02060, which is an ortholog to BcNep1, caused necrosis in all plant species tested. Conclusion: This method provides opportunities for screening Prunus germplasm with Monilinia effector proteins, to serve as a tool for identifying genetic loci that confer susceptibility to brown rot disease. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Monilinia spp. is the main pathogen that affects stone fruit, causing significant production losses, especially in seasons with favorable climatic conditions for disease development. Currently, the standard practices for controlling this disease are by means of spray programs of synthetic fungicides. Fungicide applications using treatment schedules imply an increase in the number of applications; however, the applications are justified considering the real risk of Monilinia spp. infection. Consequently, fruit surface contains a higher number and concentration of residues, but not better control of the disease. From previous studies, the epidemiology of Monilinia spp. was deeply studied in one of the main stone fruit regions of Europe, the 'Valle del Ebro', and an epidemiological model was developed to describe the brown rot epidemic pattern in this area. After that, a warning system for fungicide applications in the field was elaborated that included the main factors to be considered as fruit susceptibility, the presence of inoculum in the field, and climatological factors (temperature, leaf wetness, rainfall, or their interaction). In the present study, we present data of the warning system validation during six seasons in 38 fields of peaches and nectarines of the 'Valle del Ebro'. The results indicated that the incidence of disease caused by Monilinia spp., recorded in the field and postharvest, was similar in both plot evaluations and the calendar and warning systems. However, the disease level was higher in late varieties (3.2% and 9.3% of infected fruit recorded in the field and in postharvest, respectively) in comparison with earlier varieties (0.6% and 3.1% of infected fruit recorded in the field and in postharvest, respectively). In general, the strategy applied (the calendar or warning system) did not affect the disease level recorded. However, when fungicide treatments were applied following the warning system, the treatment reductions were higher than 50% in 96% of the trials in early varieties; meanwhile, in late varieties, this level of reduction was obtained in 77% of trials. Our data encourage the use of the proposed warning system as an effective strategy to control Monilinia spp. in peaches and nectarines, reducing the number of chemical treatments applied in the field with a high level of efficacy. [ABSTRACT FROM AUTHOR]

The development of brown rot caused by the necrotrophic fungi Monilinia spp. in stone fruit under field and postharvest conditions depends, among others, on environmental factors. The effect of temperature and humidity are well studied but there is little information on the role of light in disease development. Herein, we studied the effect of two lighting treatments and a control condition (darkness) on: (i) several growth parameters of two Monilinia spp. (M. laxa and M. fructicola) grown in vitro and (ii) the light effect in their capacity to rot the fruit (nectarines) when exposed to the different lighting treatments. We also assessed the effect of such abiotic factors in the development of the disease on inoculated nectarines during postharvest storage. Evaluations also included testing the effect of fruit bagging on disease development as well as on ethylene production. Under in vitro conditions, lighting treatments altered colony morphology and conidiation of M. laxa but this effect was less acute in M. fructicola. Such light-induced changes under in vitro development also altered the capacity of M. laxa and M. fructicola to infect nectarines, with M. laxa becoming less virulent. The performance of Monilinia spp. exposed to treatments was also determined in vivo by inoculating four bagged or unbagged nectarine cultivars, indicating an impaired disease progression. Incidence and lesion diameter of fruit exposed to the different lighting treatments during postharvest showed that the effect of the light was intrinsic to the nectarine cultivar but also Monilinia spp. dependent. While lighting treatments reduced M. laxa incidence, they enhanced M. fructicola development. Preharvest conditions such as fruit bagging also impaired the ethylene production of inoculated fruit, which was mainly altered by M. laxa and M. fructicola , while the bag and light effects were meaningless. Thus, we provide several indications of how lighting treatments significantly alter Monilinia spp. behavior both in vitro and during the interaction with stone fruit. This study highlights the importance of modulating the lighting environment as a potential strategy to minimize brown rot development on stone fruit and to extent the shelf-life period of fruit in postharvest, market, and consumer's house. [ABSTRACT FROM AUTHOR]

BACKGROUND: Brown rot caused by Monilinia spp. is the most significant disease of stone fruit. New approaches to fruit production have necessitated the development of control strategies that are more eco‐ and consumer‐friendly. An efficient field strategy to control brown rot was previously designed based on the application of two biocontrol agents (BCAs), Bacillus amyloliquefaciens CPA‐8 (CPA‐8) or Penicillium frequentans 909 (Pf909), with calendar‐based treatment. In the present study, the strategy was validated on different stone fruit hosts in four producing countries over two seasons. RESULTS: The results obtained were reported according to three different scenarios: Scenario 1, in which there was no presence of disease in the field; Scenario 2, in which high disease pressure occurred in the field and treatments (biologicals or chemicals) were not effective; and Scenario 3, with low or medium to high disease presence. The results were successful because, in general, BCA strategies were shown to control brown rot to a similar extent as chemicals strategies. We found that most of the trials conducted in this study were classed under Scenario 3 (62.5%), with only 12.5% and 25% of the trials classed under Scenarios 1 and 2, respectively. CONCLUSION: These novel findings allowed the formulation of CPA‐8 and Pf909 as valuable tools for farmers to produce stone fruits more competitively and meet consumer demand for safer and more environmentally friendly products. © 2021 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

The capacity of dispersion, persistence, and stability from biocontrol agents is essential before these organisms can be developed into a commercial product. Interactions that microorganisms establish with stone fruit trees may be beneficial in the exploitation of trees in agriculture as crop production. The natural background levels of Penicillium frequentans strain 909 dispersion, persistence, and stability were assessed after tree applications and postharvest conditions. A fingerprinting-based approach to trace genetic stability of P. frequentans along stored time and its release in the field was developed. P. frequentans was successfully traced and discriminated. This strain was dispersed well in treated trees, persisting in the ecosystem up to 2 weeks and staying genetically stable after 36 months of storage. However, the dispersal of P. frequentans was very limited on around untreated trees and soil. P. frequentans dispersed randomly into the air, and its presence reduced from the first day to disappear completely at 15–21 days after application. Great losses of P. frequentans and its increased dispersal in open field conditions probably resulted from rainfall. Biological control strategies with Pf909 were discussed. [ABSTRACT FROM AUTHOR]

Infections by the fungus Monilinia laxa, the main cause of brown rot in Europe, result in considerable losses of stone fruit. Herein, we present a comprehensive transcriptomic approach to unravel strategies deployed by nectarine fruit and M. laxa during their interaction. We used M. laxa-inoculated immature and mature fruit, which was resistant and susceptible to brown rot, respectively, to perform a dual RNA-Seq analysis. In immature fruit, host responses, pathogen biomass, and pathogen transcriptional activity peaked at 14–24 h post inoculation (hpi), at which point M. laxa appeared to switch its transcriptional response to either quiescence or death. Mature fruit experienced an exponential increase in host and pathogen activity beginning at 6 hpi. Functional analyses in both host and pathogen highlighted differences in stage-dependent strategies. For example, in immature fruit, M. laxa unsuccessfully employed carbohydrate-active enzymes (CAZymes) for penetration, which the fruit was able to combat with tightly regulated hormone responses and an oxidative burst that challenged the pathogen's survival at later time points. In contrast, in mature fruit, M. laxa was more dependent on proteolytic effectors than CAZymes, and was able to invest in filamentous growth early during the interaction. Hormone analyses of mature fruit infected with M. laxa indicated that, while jasmonic acid activity was likely useful for defense, high ethylene activity may have promoted susceptibility through the induction of ripening processes. Lastly, we identified M. laxa genes that were highly induced in both quiescent and active infections and may serve as targets for control of brown rot. [ABSTRACT FROM AUTHOR]

The microbial variability on the host plant surface must be maintained because population diversity and quantity are essential to avoid disease development. It would be necessary to examine the patterns and mechanisms associated with the massive and reiterative introduction of a microbial pest control agent. The effect of inundative releases of biopesticide formulations containing Penicillium frequentans for the control of Monilinia spp. populations, and the effect on fruit surface microbiota on 18 stone fruit field experiments located in four European countries for more than two crop seasons against brown rot were studied. P. frequentans was monitored after application in order to assess whether it was persistent or not in the environment. Hydrolysis of fluorescein diacetate and denaturing gradient gel electrophoresis were used to study the effects of P. frequentans on fungal and bacterial non-target populations on fruit surface. The effect of P. frequentans formulations on the populations of Monilinia spp. on fruit was also assessed in different orchards. P. frequentans population on stone fruit surfaces showed ranged from 100 to 10,000 CFU cm−2, and postharvest recovered populations were more than 10–100-fold higher than preharvest recovered populations. The population of P. frequentans varied among orchards and years, rather than by the type of formulation. P. frequentans formulation reduced Monilinia spp. population and brown rot and latent infections caused by this pathogen both before and at harvest, while stabilizing or increasing antagonist populations and avoiding non-target microorganisms. However, fungicides reduced significantly the microbial activity on nectarine surfaces. • Pf909 was effective with no harmful effect on peach microbiota balance. • Fungicides used against brown rot reduced significantly the microbial activity on stone fruit surfaces. • Populations of Pf909 after application on fruit did not persistently exceed the natural background level of the species. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • No synergistic effect was obtained with the joint application of P. frequentans and B. amyloliquefaciens. • B. amyloliquefaciens inhibited the germination and growth of P. frequentans. • B. amyloliquefaciens also competes with P. frequentans once applied on fruit surfaces. • Most commercial fungicides and insecticides were compatible with P. frequentans in a brown rot integrated management. Abstract Background To improve efficacies achieved through biocontrol agent application, there has been increasing interest in studying the efficacy of biocontrol agent with other existing technologies like chemicals or different biocontrol agent combinations. The compatibility of Penicillium frequentans (Pf909) with modern inputs in plant protection like fungicides (chemicals and biologicals) and insecticides is a pre-requisite for developing integrated brown rot management strategies. Results The combination of P. frequentans and Bacillus amyloliquefaciens , each with different mechanisms of action, competition and antibiosis, respectively, did not improve the biocontrol of brown rot when each treatment was applied individually. P. frequent ans and B. amyloliquefaciens could not be combined together in the same tank, because bacteria inhibited the germination and growth of P. frequentans tubes in suspension. Furthermore, B. amyloliquefaciens also competes with P. frequentans once applied on fruit surfaces. Additionally, bacteria reduced the growth of P. frequentans CFUs on media, at different 25 °C and 30 °C throughout the incubation periods. However, 76% of fungicides and 90% of insecticides commonly used against stone fruit pests are compatible with P. frequentans. Conclusions Results indicated that the most commercial chemical pesticides could be simultaneously applied with Pf909 in integrated pest control, but not B. amyloliquefaciens. [ABSTRACT FROM AUTHOR]

The most devastating fungal disease of peaches and nectarines is brown rot, caused by Monilinia spp. Among the many plant responses against biotic stress, plant terpenoids play essential protective functions, including antioxidant activities and inhibition of pathogen growth. Herein, we aimed to characterize the expression of terpenoid biosynthetic genes in fruit tissues that presented different susceptibility to brown rot. For that, we performed artificial inoculations with Monilinia laxa at two developmental stages (immature and mature fruit) of two nectarine cultivars ('Venus' –mid-early season cultivar - and 'Albared' –late season cultivar-) and in vitro tests of the key compounds observed in the transcriptional results. All fruit were susceptible to M. laxa except for immature 'Venus' nectarines. In response to the pathogen, the mevalonic acid (MVA) pathway of the 'Venus' cultivar was highly induced in both stages rather than the methylerythritol phosphate (MEP) pathway, being the expression of some MEP-related biosynthetic genes [e.g., PROTEIN FARNESYLTRANSFERASE (PpPFT), and 3S-LINALOOL SYNTHASE (PpLIS)] different between stages. In 'Albared', both stages presented similar responses to M. laxa for both pathways. Comparisons between cultivars showed that HYDROXYMETHYLGLUTARYL-CoA REDUCTASE (PpHMGR1) expression levels were common in susceptible tissues. Within all the terpenoid biosynthetic pathway, linalool- and farnesal-related pathways stood out for being upregulated only in resistant tissues, which suggest their role in mediating the resistance to M. laxa. The in vitro antifungal activity of linalool and farnesol (precursor of farnesal) revealed fungicidal and fungistatic activities against M. laxa , respectively, depending on the concentration tested. Understanding the different responses between resistant and susceptible tissues could be further considered for breeding or developing new strategies to control brown rot in stone fruit. • Monilinia laxa differentially infects immature fruit of two nectarine cultivars. • Terpenoid biosynthetic gene expression is impaired in response to brown rot. • MVA pathway is induced rather than MEP pathway in M. laxa-response. • Farnesal- and linalool-related genes are upregulated only in M. laxa-resistant tissues. • Linalool and farnesol (precursor of farnesal) have antifungal activity against M. laxa in vitro. [ABSTRACT FROM AUTHOR]

Fruit volatile organic compounds (VOCs) can be emitted by stone fruit in response to biotic stress. One of the main fungal diseases of stone fruit is brown rot, caused by species of Monilinia spp. Hence, we aimed to reveal the most relevant VOCs that participate either in resistance or susceptibility to Monilinia laxa in nectarines. To this aim, we analyzed the VOCs emitted by two developmental stages (immature, one month before harvest; mature, commercial harvest) of two nectarine cultivars, with different susceptibility to M. laxa. Furthermore, we also determined the VOCs profile of M. laxa grown in in vitro culture media based on peach juice. Results elucidated 34 VOCs whose production pattern was different among samples (control and inoculated of both stages and cultivars), being 13 VOCs also emitted by M. laxa culture. A hierarchical analysis and a multivariate analysis exhibited the variations in the VOCs profile of all samples according to their susceptibility to M. laxa , and the suitability of the model to predict the M. laxa disease (91.94% of the total variation). In general, results highlighted i) a group of VOCs, positively correlated with M. laxa disease, that were emitted by visual M. laxa symptomatic tissues (e.g. , aldehyde (E,E)− 2,6-nonadienal) and also by M. laxa itself (e.g. , terpenoids alpha-muurolene and (E)-beta-ionone), and ii) a group of VOCs, negatively correlated with brown rot disease, that were emitted by tissues with no visual M. laxa symptoms (e.g. , ketone butyrolactone and aldehyde (E)− 2-decenal) and also by M. laxa itself (e.g. , aldehyde decanal), suggesting an antifungal role of these compounds. Therefore, this study provides putative potential VOCs that not only will help to improve the knowledge of brown rot development on nectarines, but also provides target volatiles that may serve as potential brown rot control compounds. • Profile of volatile organic compounds (VOCs) is related to susceptibility to M. laxa. • Some M. laxa -emitted VOCs are the same as those produced by the nectarine- M. laxa pathosystem. • Fruit (E,E)− 2,6-nonadienal may favor the host susceptibility to M. laxa. • Alpha-muurolene could be produced by the pathogen as a virulence factor. • Among others, (E)− 2-decenal and butyrolactone could be antifungal compounds. [ABSTRACT FROM AUTHOR]

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 postharvest. • M. laxa and M. fructicola present a similar behaviour, but not M. fructigena. • The conidial production is higher in blue light for M. laxa and M. fructicola. • M. fructicola growth rate is reduced under red light compared to the other species. • Virulence in nectarines decreases in M. laxa and M. fructicola under black light. • M. fructigena reveals a similar behaviour among darkness and light wavelengths. [ABSTRACT FROM AUTHOR]

[Display omitted] • Phylogenetic analysis differentiated stone and pome fruit sirtuin protein sequences. • Differential inter and intra species (apple, pear and nectarine) gene expression profile of SRTs. • PbSRT1 is negatively correlated with the pear hormones ABA, IAA, GA1 and zeatin. • PbSRT2 is associated to the ripening process in an ethylene-independent manner. • Sugar accumulation during pear ripening may be mediated by PbSRT2. Epigenetic regulation is crucial to ensure a coordinated control of the different events that occur during fruit development and ripening. Sirtuins are NAD+-dependent histone deacetylases involved in the regulation of gene expression of many biological processes. However, their implications in the Rosaceae family remains unexplored. Accordingly, in this work, we demonstrated the phylogenetic divergence of both sirtuins among Rosaceae species. We then characterized the expression pattern of both SRT1 and SRT2 in selected pome and stone fruit species. Both SRT1 and SRT2 significantly changed during the fruit development and ripening of apple, nectarine and pear fruit, displaying a different expression profile. Such differences could explain in part their different ripening behaviour. To further unravel the role of sirtuins on the fruit development and ripening processes, a deeper analysis was performed using pear as a fruit model. In pear, PbSRT1 gene expression levels were negatively correlated with specific hormones (i.e. abscisic acid, indole-3-acetic acid, gibberellin A1 and zeatin) during the first phases of fruit development. PbSRT2 seemed to directly mediate pear ripening in an ethylene-independent manner. This hypothesis was further reinforced by treating the fruit with the ethylene inhibitor 1-methylcyclopropene (1-MCP). Instead, enhanced PbSRT2 along pear growth/ripening positively correlated with the accumulation of major sugars (R2 > 0.94), reinforcing the idea that sugar metabolism may be a target of epigenetic modifications during fruit ripening. Overall, the results from this study point out, for the first time, the importance that sirtuins have in the regulation of fruit growth and ripening of pear fruit by likely regulating hormonal and sugar metabolism. [ABSTRACT FROM AUTHOR]

• Host ethylene production differentially affects M. laxa and M. fructicola infection capacity. • 1-MCP and exogenous ethylene treatments enhance peach petal ethylene production. • Peach petals present different ability to cope with both M. laxa and M. fructicola. • M. laxa but not M. fructicola inhibits host ethylene production. • Host ERFs gene expression is differentially modulated by both M. laxa and M. fructicola. Monilinia spp. may infect stone fruit at any growth stage, although susceptibility to brown rot depends on both host properties and climatological conditions. This said, no studies deciphering the host response in the interaction between peach blossoms and Monilinia spp. are yet available. This study presents an in-depth characterization of the role of ethylene in the interaction of 'Merrill O'Henry' peach petals (Prunus persica (L.) Batch) with Monilinia laxa and M. fructicola. We investigated the physiological responses of the host and the fungi to the application of ethylene and 1-methylcyclopropene (1-MCP) as well as the molecular patterns associated with the biosynthetic and ethylene-dependent responses during the interaction of both Monilinia species with the host. The incidence of both species was differentially affected by 1-MCP and ethylene; M. laxa was favoured by the enhanced host ethylene production associated with the treatments whereas M. fructicola reduced its infection capacity. Such differences were host-dependent as treatments did not affect growth or colony morphology of Monilinia spp. Besides, host ethylene production was altered in M. laxa inoculated petals, either by the fungus or the host itself. Molecular analysis revealed some important ERFs that could be involved in the different ability of both species to activate a cascade response of peach petals against these pathogens. [ABSTRACT FROM AUTHOR]