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.

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.

Transcriptional profiling of the terpenoid biosynthesis pathway and in vitro tests reveal putative roles of linalool and farnesal in nectarine resistance against brown rot.

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.

Non-volatile signals and redox mechanisms are required for the responses of Arabidopsis roots to Pseudomonas oryzihabitans.

Soil bacteria promote plant growth and protect against environmental stresses, but the mechanisms involved remain poorly characterized, particularly when there is no direct contact between the roots and bacteria. Here, we explored the effects of Pseudomonas oryzihabitans PGP01 on the root system architecture (RSA) in Arabidopsis thaliana seedlings. Significant increases in lateral root (LR) density were observed when seedlings were grown in the presence of P. oryzihabitans, as well as an increased abundance of transcripts associated with altered nutrient transport and phytohormone responses. However, no bacterial transcripts were detected on the root samples by RNAseq analysis, demonstrating that the bacteria do not colonize the roots. Separating the agar containing bacteria from the seedlings prevented the bacteria-induced changes in RSA. Bacteria-induced changes in RSA were absent from mutants defective in ethylene response factor (ERF109), glutathione synthesis (pad2-1, cad2-1, and rax1-1) and in strigolactone synthesis (max3-9 and max4-1) or signalling (max2-3). However, the P. oryzihabitans-induced changes in RSA were similar in the low ascorbate mutants (vtc2-1and vtc2-2) to the wild-type controls. Taken together, these results demonstrate the importance of non-volatile signals and redox mechanisms in the root architecture regulation that occurs following long-distance perception of P. oryzihabitans.

Environmentally driven transcriptomic and metabolic changes leading to color differences in "Golden Reinders" apples.

Apple is characterized by its high adaptation to diverse growing environments. However, little is still known about how different environments can regulate at the metabolic or molecular level specific apple quality traits such as the yellow fruit peel color. In this study, changes in carotenoids and chlorophylls, antioxidants as well as differences in the transcriptome were investigated by comparing the peel of "Golden Reinders" apples grown at different valley and mountain orchards. Mountain environment favored the development of yellow color, which was not caused by an enhanced accumulation of carotenoids but rather by a decrease in the chlorophyll content. The yellow phenotype was also associated to higher expression of genes related to chloroplast functions and oxidative stress. Time-course analysis over the last stages of apple development and ripening, in fruit from both locations, further revealed that the environment differentially modulated isoprenoids and phenylpropanoid metabolism and pointed out a key role for H2O2 in triggering apple peel degreening. Overall, the results presented herein provide new insights into how different environmental conditions regulate pigment and antioxidant metabolism in apple leading to noticeable differences in the apple peel color.

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 postharvest.

Using plant growth-promoting microorganisms (PGPMs) to improve plant development under in vitro culture conditions.

MAIN CONCLUSION: The use of beneficial microorganisms improves the performance of in vitro - cultured plants through the improvement of plant nutrition, the biological control of microbial pathogens or the production of phytohormones that promote plant growth and development. Plant in vitro culture techniques are highly useful to obtain significant amounts of true-to-type and disease-free plant materials. One of these techniques is clonal micropropagation which consists on the establishment of shoot tip cultures, shoot multiplication, in vitro rooting and acclimatization to ex vitro conditions. However, in some cases, the existence of recalcitrant genotypes, with a compromised multiplication and rooting ability, or the difficulties to overcome the overgrowth of endophytic contaminations might seriously limit its efficiency. In this sense, the establishment of beneficial interactions between plants and plant growth-promoting microorganisms (PGPMs) under in vitro culture conditions might represent a valuable approach to efficiently solve those restrictions. During the last years, significant evidence reporting the use of beneficial microorganisms to improve the yield of in vitro multiplication or rooting as well as their acclimatization to greenhouse or soil conditions have been provided. Most of these positive effects are strongly linked to the ability of these microorganisms to provide in vitro plants with nutrients such as nitrogen or phosphorous, to produce plant growth regulators, to control the growth of pathogens or to mitigate stress conditions. The culture of A. thaliana under aseptic conditions has provided high-quality knowledge on the root development signaling pathways, involving hormones, triggered in the presence of PGPMs. Overall, the present article offers a brief overview of the use of microorganisms to improve in vitro plant performance during the in vitro micropropagation stages, as well as the main mechanisms of plant growth promotion associated with these microorganisms.

ROS-scavenging-associated transcriptional and biochemical shifts during nectarine fruit development and ripening.

ROS are known as toxic by-products but also as important signaling molecules playing a key role in fruit development and ripening. To counteract the negative effects of ROS, plants and fruit own multiple ROS-scavenging mechanisms aiming to ensure a balanced ROS homeostasis. In the present study, changes in specific ROS (i.e. H2O2) as well as enzymatic (SOD, CAT, POX, APX) and non-enzymatic (phenylpropanoids, carotenoids and ascorbate) ROS-scavenging systems were investigated along four different stages of nectarine (cv. 'Diamond Ray') fruit development and ripening (39, 70, 94 and 121 DAFB) both at the metabolic (28 individual metabolites or enzymes) and transcriptional level (24 genes). Overall, our results demonstrate a complex ROS-related transcriptome and metabolome reprogramming during fruit development and ripening. At earlier fruit developmental stages an increase on the respiration rate is likely triggering an oxidative burst and resulting in the activation of specific ethylene response factors (ERF1). In turn, ROS-responsive genes or the biosynthesis of specific antioxidant compounds (i.e. phenylpropanoids) were highly expressed or accumulated at earlier fruit developmental stages (39-70 DAFB). Nonetheless, as the fruit develops, the decrease in the fruit respiration rate and the reduction of ERF1 genes leads to lower levels of most non-enzymatic antioxidants and higher accumulation of H2O2. Based on available literature and the observed accumulation dynamics of H2O2, it is anticipated that this compound may not only be a by-product of ROS-scavenging but also a signaling molecule accumulated during the ripening of nectarine fruit.

Optimization of a food industry-waste-based medium for the production of the plant growth promoting microorganism Pseudomonas oryzihabitans PGP01 based on agro-food industries by-products.

In this study, three wastes based on potato peels and pulps, tomato seeds and wheat bran were used as basis for the preparation of a cheap medium to produce the bacterium P. oryzihabitans PGP01. In flasks experiments, P. oryzihabitans PGP01 growth at 25 °C in a medium based on frozen potato peels and pulp (FPP) with tryptone as a nitrogen source resulted in the maximum production compared to the commercial TSB medium. In the scale-up to 2 L bioreactors, FPP supplemented with tryptone, molasses, NaCl and K2HPO4 allowed to reach similar biomass production than in the TSB medium. A maximum growth of 4.4 × 109 CFU mL-1 after setting the agitation and the air flux conditions at 400 rpm and 0.75 vvm. Finally, P. oryzihabitans PGP01 growing in this optimized medium conserved its biological activity showing the expected effect in root development previously reported for this microorganism.

Light Intensity Alters the Behavior of Monilinia spp. in vitro and the Disease Development on Stone Fruit-Pathogen Interaction.

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.

PbSRT1 and PbSRT2 regulate pear growth and ripening yet displaying a species-specific regulation in comparison to other Rosaceae spp.

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.

Rhizosphere microorganisms enhance in vitro root and plantlet development of Pyrus and Prunus rootstocks.

MAIN CONCLUSION: The in vitro application of rhizosphere microorganisms led to a higher rooting percentage in Pyrus Py12 rootstocks and increased plant growth of Pyrus Py170 and Prunus RP-20. The rooting of fruit tree rootstocks is the most challenging step of the in vitro propagation process. The use of rhizosphere microorganisms to promote in vitro rooting and plant growth as an alternative to the addition of chemical hormones to culture media is proposed in the present study. Explants from two Pyrus (Py170 and Py12) rootstocks and the Prunus RP-20 rootstock were inoculated with Pseudomonas oryzihabitans PGP01, Cladosporium ramotenellum PGP02 and Phoma sp. PGP03 following two different methods to determine their effects on in vitro rooting and plantlet growth. The effects of the microorganisms on the growth of fully developed Py170 and RP-20 plantlets were also studied in vitro. All experiments were conducted using vermiculite to simulate a soil system in vitro. When applied to Py12 shoots, which is a hard-to-root plant material, both C. ramotenellum PGP02 and Phoma sp. PGP03 fungi were able to increase the rooting percentage from 56.25% to 100% following auxin indole-3-butyric acid (IBA) treatment. Thus, the presence of these microorganisms clearly improved root development, inducing a higher number of roots and causing shorter roots. Better overall growth and improved stem growth of treated plants was observed when auxin treatment was replaced by co-culture with microorganisms. A root growth-promoting effect was observed on RP-20 plantlets after inoculation with C. ramotenellum PGP02, while P. oryzihabitans PGP01 increased root numbers for both Py170 and RP-20 and increased root growth over stem growth for RP-20. It was also shown that the three microorganisms P. oryzihabitans PGP01, C. ramotenellum PGP02 and Phoma sp. PGP03 were able to naturally produce auxin, including indole-3-acetic acid (IAA), at different levels. Overall, our results demonstrate that the microorganisms P. oryzihabitans PGP01 and C. ramotenellum PGP02 had beneficial effects on in vitro rooting and plantlet growth and could be applied to in vitro tissue culture as a substitute for IBA.

Global analysis of the apple fruit microbiome: are all apples the same?

We present the first worldwide study on the apple (Malus × domestica) fruit microbiome that examines questions regarding the composition and the assembly of microbial communities on and in apple fruit. Results revealed that the composition and structure of the fungal and bacterial communities associated with apple fruit vary and are highly dependent on geographical location. The study also confirmed that the spatial variation in the fungal and bacterial composition of different fruit tissues exists at a global level. Fungal diversity varied significantly in fruit harvested in different geographical locations and suggests a potential link between location and the type and rate of postharvest diseases that develop in each country. The global core microbiome of apple fruit was represented by several beneficial microbial taxa and accounted for a large fraction of the fruit microbial community. The study provides foundational information about the apple fruit microbiome that can be utilized for the development of novel approaches for the management of fruit quality and safety, as well as for reducing losses due to the establishment and proliferation of postharvest pathogens. It also lays the groundwork for studying the complex microbial interactions that occur on apple fruit surfaces.

Field validation of biocontrol strategies to control brown rot on stone fruit in several European countries.

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.

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.

Depicting the battle between nectarine and Monilinia laxa: the fruit developmental stage dictates the effectiveness of the host defenses and the pathogen's infection strategies.

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.

Ethylene biosynthesis and response factors are differentially modulated during the interaction of peach petals with Monilinia laxa or Monilinia 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.

Balance between resilient fruit surface microbial community and population of Monilinia spp. after biopesticide field applications of Penicillium frequentans.

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.

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.

Dispersion, persistence, and stability of the biocontrol agent Penicillium frequentans strain 909 after stone fruit tree applications.

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.