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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.

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.

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.

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.

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.

[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. frequentans 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.

[EN] Ampelomyces quisqualis CPA-9 was reported as a promising biocontrol fungus against powdery mildew on cucurbits, although its formulation had not been developed. The present work aimed to develop a fluidised-bed spray-dried formulation of CPA-9 that included film-forming compounds, which could improve the behaviour of the microorganism under practical conditions. Film forming compounds, efficacy, shelf life, physical stability as a function of aw, and solubility in water were determined. A film-forming formulation based on native potato starch and pregelatinised potato starch as carrier and binder was obtained, and sucrose plus skimmed milk were used as protective compounds. Dehydrated conidia maintained the efficacy of fresh cells against Podosphaera xanthii on zucchini leaves, and powdery mildew was significantly reduced compared with control treatment. Despite of results obtained for the glassy state guaranteed the stability of the powder against sticking and caking processes, the shelf life of the product was limited to 4 months of storage at 4 degrees C. On the other hand, the formulation was mainly dispersible, due to the high concentration of native potato starch which reduces the solubility of the product. Results obtained in this work, such as the high efficacy of the developed product, the low cost of the employed substances, the film-forming ability of the rehydrated powder, and finally the effectiveness of the dehydration process, confirmed the potential of the product. Further studies to verify the improvement of CPA-9 behaviour under practical conditions or to improve the storage conditions to enlarge the shelf life of the BCA should be done to consider the developed product as a biocontrol-based product for cucurbits against powdery mildew.

Different treatments based on the biocontrol agent (BCA) Bacillus amyloliquefaciens CPA-8 to control brown rot under field conditions were evaluated as alternative to chemical applications. As part of a well-designed disease program that enables the integration of BCAs into cropping systems, testing of the sensitivity of Monilinia laxa and Monilinia fructicola at different doses of CPA-8 were conducted in stone fruit under laboratory conditions. CPA-8 dose of 107 CFU mL−1 reduced more than 60.0 and 75.5% of brown rot incidence and severity, respectively. Once in the orchard, different degree of biocontrol activity was obtained depending on the inoculum pressure, which was mainly associated with meteorological conditions. Under drastic disease pressure, neither CPA-8 treatment nor the chemicals controlled the disease at harvest and only the chemical treatment reduced postharvest brown rot incidence. However, when Monilinia spp. incidence was close to the standard levels recorded in the area, treatments based on CPA-8 formulations proved to be efficacious. At harvest, BA3, BA4 treatments (CPA-8 optimised products) and PF + BA3 treatment (Penicillium frequentans combined with CPA- 8) reduced Monilinia spp. incidence compared to the control (54.7–64.1%) and similar to the chemicals. At postharvest, almost all CPA-8-based treatments (except PF + BA3) controlled the pathogen with BA4 treatment being as much effective as the chemicals (50.3% of disease reduction). Finally, the population dynamics of CPA-8 on treated fruit surface remained after treatment application, at harvest and at postharvest shelf-life (>104 CFU cm−2). This study highlights the potential of B. amyloliquefaciens CPA-8 as alternative or complementary strategies to control Monilinia spp. © 2017 Elsevier Ltd

This is the peer reviewed version of the following article: Carbó, A., Torres, R., Usall, J., Marín, A., Chiralt, A. and Teixidó, N. (2019), Novel film¿forming formulations of the biocontrol agent Candida sake CPA¿1: biocontrol efficacy and performance at field conditions in organic wine grapes. Pest. Manag. Sci., 75: 959-968, which has been published in final form at https://doi.org/10.1002/ps.5200. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving., [EN] BACKGROUND The biocontrol agent (BCA) Candida sake CPA-1 has previously effectively reduced Botrytis bunch rot (BBR) and it was also suggested as a promising strategy to control sour rot in grapes under field conditions. However, biocontrol efficacy of solid formulations of CPA-1 has never been tested in field trials. The present study aims to confirm the efficacy against BBR and sour rot in grapes under field conditions of two novel formulations recently developed by the addition of biodegradable coatings using a fluidized-bed spray-drying system. RESULTS Novel film-forming formulations of the BCA C. sake CPA-1 controlled B. cinerea as well as liquid formulation. Sour rot control resulted better in the second season and severity reductions were more satisfactory than incidence control. Visual and cryoSEM observations revealed that film-forming treatments were uniformly distributed on plant surfaces. CPA-1 coating could be observed on grapes at harvest time. CONCLUSION The results of this work suggest that solid formulations would be a competitive alternative to conventional fungicides because they were easy to package and transport, and cell viability could be maintained for a long period of time.

[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.

[EN] Multiple formulations of known biocontrol agent (BCA) Candida sake, containing different coatingforming polymers and surfactants were tested at different polymer:BCA ratios, in order to improve control of Botrytis cinerea on grapes. The BCA cell viability on the grape surface was analyzed and reduction in disease incidence and severity was determined. Coating-forming solids improved the survival and effi- cacy of C. sake as a BCA against B. cinerea, depending on the polymer type and ratio. The incorporation of surfactants did not improve survival or disease control, although they promoted a better cell dispersion on the grape surface. Cell growth of the antagonist during incubation led to the formation of aggregates, even when surfactants were present. Sodium caseinate and starch were the most suitable polymers to formulate C. sake preparations to obtain coating-forming systems with this BCA and to increase its survival and efficacy at the minimum economic cost of the ingredients. 2016 Elsevier Inc. All rights reserved