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Abiotic factors such as light influence the physicochemical properties of fruit and may alter the response of the fruit to the environment. The aim of this study was to investigate the effect of two postharvest storage conditions on the overall quality and natural fungal disease incidence (fruit decay) of organic nectarines. Experiments were conducted with four organically grown nectarine cultivars (two early-mid season and two late-season) that were unbagged or bagged during preharvest. After harvest, they were stored for 7–9 days in darkness or under a treatment with lighting. Quality parameters (weight, diameter, firmness, soluble solids content, titratable acidity, and single index of absorbance difference), ethylene production, and fruit decay (% incidence of decay) were evaluated. Preharvest bagging reduced fruit decay in the late-season cultivars, for which storage under darkness reduced fungal decay (up to 100%) more than storage under lighting treatment (47% reduction). Bagging altered the initial fruit quality, but values were within official recommendations. Storage conditions reduced differences attributed to bagging, especially under storage with lighting. This work highlighted the importance of modulating the light, both in the field by fruit bagging and during postharvest, to reduce fruit decay and improve fruit quality. These results may serve as a tool for both farmers and postharvest chain managers. info:eu-repo/semantics/acceptedVersion

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 info:eu-repo/semantics/acceptedVersion

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.

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.

It is already well known that ethylene plays a crucial role in peach fruit growth and ripening, by triggering an unset of biochemical and physiological changes that finally make the fruit attractive for consumption. This said, ethylene is not only responsible for fruit ripening but, in conjunction with other hormones, or key compounds (ROS, polyamines, etc.) is involved in the plant response to numerous abiotic stresses (drought, salt and heat tolerance) as well as the plant/fruit response against certain pathogens. Among peaches, one of the most devastating pathogens is the brown rot causing fungus Monilinia spp. that can affect the fruit both on the field or postharvest. Nonetheless scarce information exists regarding the Monilinia-peach interaction from a physiological and molecular perspective. In this sense, recent studies point out to the importance of ethylene during such interaction, which seems to be dependent on the fruit developmental stage and also on the Monilinia species or even the strain's virulence. Why the fruit or the fungus reacts different to distinct Monilinia species or strains and why such reaction depends on the fruit physiological stage is, however, still elusive. Accordingly, this review aims to shed light on the role of ethylene, alone or through a complex cross-talk with other compounds, not only during peach development and ripening but also during the Monilinia-peach interaction. Based on the available literature, it is clear that not only ethylene biosynthesis but ethylene signaling and the activation of ethylene response factors via ROS may play an essential role during this specific host-pathogen interaction. info:eu-repo/semantics/acceptedVersion

An autocatalytic burst in ethylene production generally accompanies ripening of detached apple fruit. However, if apples are left to ripen attached to the tree, some cultivars, will not experience this autocatalytic ethylene production and the fruit will never acquire the organoleptic properties of detached ripened fruit. Accordingly, the present study aimed to understand how the hormonal crosstalk regulates ripening in ‘Golden Reinders’ apples, ripened on-tree, detached from the tree or after a period of cold storage, following the commercial harvest date. Our results showed that during on-tree ripening, ethylene production remained low, and no significant changes were observed in fruit colour or firmness. In fruit ripened detached from the tree, ethylene production was preceded by an increase of indole 3-acetic acid (IAA) and gibberellic acid (GA3) levels, whereas a cold-induced accumulation of jasmonic acid (JA) seemed to induce an earlier initiation of the climacteric burst in fruit ripened after cold storage. In both postharvest conditions, the accumulation of abscisic acid (ABA) may be either needed or facilitating the action of ethylene on triggering some ripening-related changes, pointing out the importance of both hormones in mediating apple ripening. In contrast, changes in sugars and ROS/antioxidants did not vary among the different ripening scenarios suggesting that none of the measured compounds may act as signalling molecules during apple ripening. Collectively, our results highlight that ethylene together with ABA played a crucial role in triggering ripening-related changes during postharvest ripening of ‘Golden Reinders’ apples. info:eu-repo/semantics/acceptedVersion

[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., This research was supported by the INIA (Instituto Nacional de Investigacion y Tecnologia Agraria y Alimentaria) and FEDER (Fondo Europeo de Desarrollo Regional) through national project RTA201200067C0201. The research was also supported by INIA and FSE (Fondo Social Europeo) for the PhD grant awarded to A. Carbo. The authors are also grateful to the CERCA Programme/Generalitat de Catalunya and the technical support provided by Cristina Solsona, Celia Sanchez and Andrea Berge.

The use of the GreenTray® TIS bioreactor for the in vitro analysis of the interaction between plantlets and two Plant Growth-Promoting Microorganisms (PGPMs) (Pseudomonas oryzihabitans PGP01 and Cladosporium ramotenellum PGP02) is reported herein. This in vitro culture system improved micropropagation of Prunus Rootpac 20® rootstock, showing greater shoot length and fresh weight compared with culture in semisolid agar-containing medium. Plant responses in co-culture with the PGPMs, their ability to control endophytes growth in the culture media and hormonal changes associated to plant growth were studied in the GreenTray® culture system. Inoculation with P. oryzihabitans PGP01 had no significant effects, whereas C. ramotenellum PGP02 considerably reduced endophytes population after 5 days post inoculation. However, the fungi overgrew the plants roots seriously reducing their growth. These results suggested the effect of the medium pH as a factor to control microbial endophytic growth in the culture medium. A higher P. oryzihabitans PGP01 concentration was inoculated in culture media previously adjusted to pH 5.7 or 7, observing an increased number of roots when plantlets were co-cultured for 15 days with the bacterium in medium adjusted to pH 5.7. This was associated with changes in the levels of IAA in the culture medium, and higher leaf chlorophyll contents than in the non-inoculated cultured plantlets. P. oryzihabitans PGP01 was not able to reduce endophytes growth in either of both media pHs. Altogether, the GreenTray® bioreactor was shown as an efficient system to in vitro micropropagate RP-20 explants as well as to monitor its interaction with PGPMs. info:eu-repo/semantics/acceptedVersion

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 info:eu-repo/semantics/acceptedVersion

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/acceptedVersion

Assays were conducted to understand the effect of various factors involved with screening for resistance to Monilinia fructicola in stone fruit. First, the effect of maturity heterogeneity was determined in a set of fruit graded into three apparent maturity classes according to the IAD index for measurements taken with a DA-Meter. Second, different conidia concentrations, incubation times and M. fructicola strains were evaluated to optimize the methodology. Furthermore, the effects of fruit disinfection on M. fructicola, Penicillium expansum and Rhizopus stolonifer growth were studied. Finally, the developed methodology was applied to a set of commercial varieties of stone fruit to determine its suitability for identifying the level of susceptibility to brown rot. The results obtained indicated significant differences between wounded and non-wounded fruit, inoculum concentration and incubation time. The effect of strain aggressiveness was also confirmed, whereas, in general, no significant differences were observed among the fruit collected at the same harvest date. Our results also show that disinfection of fruit prior to inoculation had an effect on the infection process. In addition, the use of this methodology in commercial varieties of stone fruit allowed distinctions to be made among the levels of susceptibility to brown rot. Hence, the developed methodology could be applied to resistance screening in breeding programmes, as well as studying the genetic basis of brown rot resistance.

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. info:eu-repo/semantics/acceptedVersion

10 Pág. Departamento de ​Protección Vegetal (INIA), 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., This research was supported by the European project BIOCOMES FP7‐612713 and by the Secretaria d’Universitats i Recerca del Departament d’Economia i Coneixement de la Generalitat de Catalunya for the PhD grant 2014‐FI‐B00367 (Amparo M. Gotor Vila). The authors also thank CERCA Program (Generalitat de Catalunya) and the technical and logistical support provided by Amparo Gotor‐Vila, Daniel Lastrada, Cèlia Sanchez, María Sisquella, and Cristina Solsona.

‘Golden Reinders’ apple quality parameters, fruit physiology, biochemical composition and susceptibility to Penicillium expansum and Rhizopus stolonifer were analysed in fruit harvested from four different locations (two valley and two mountain orchards) and from the same valley orchard at six different maturity stages. Growing location strongly influenced the taste- and health-related fruit composition whereas the fruit maturity at harvest mainly affected the ethylene biosynthetic pathway and ethylene-dependant quality traits such as the fruit firmness and starch index. The fruit maturity at harvest, but not the growing location, also affected the severity of the infection caused by P. expansum and R. stolonifer, with mature fruit showing higher susceptibility to pathogen infection. Besides, by employing a Partial Least Square (PLS) regression model, our data showed that the severity of the lesions caused by R. stolonifer were intimately related to the fruit ethylene production. Overall, the results from this study demonstrate that differences in environmental conditions between orchards (mountain vs valley) strongly influenced the composition of ‘Golden Reinders’ apples without affecting the susceptibility of the fruit to two major postharvest pathogens. info:eu-repo/semantics/acceptedVersion

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.

Overgrowth of endophytes in some in vitro cultures may disrupt the normal shoot tip growth and proliferation, being necessary to obtain endophytes-free cultures to achieve a normal plant micropropagation process. To remove these contaminations from the culture medium, antibiotics are commonly added to the culture medium. However, its use in plant production must be urgently reduced because of the current restrictions imposed by the European Union. For that purpose, the effect of acidic low (pH 3) and neutral (pH 7) pH was tested in the GreenTray® TIS bioreactor as an alternative to control endophytes growth without affecting the micropropagation of the Prunus rootstock RP-20 explants. The results demonstrated that culture at pH 3 did not affect the number of shoots, shoot FW, shoot length and the amount of chlorophyll pigments, but significantly reduced endophytes population. The identification also revealed that Roseomonas mucosa, Microbacterium oxydans, Bacillus subtilis and Luteibacter yeojuensis were the bacterial isolates responsible of those contaminations. These results might suppose a real breakthrough in the in vitro tissue culture field, although more research is required to meet the pH requirements for the different plant species and other endophytic. info:eu-repo/semantics/acceptedVersion

Background: Apple is one of the most highly consumed fruits worldwide and is the largest fruit crop produced in temperate regions. Fruit quality, safety and long-term storage are issues that are important to growers, distributors, and consumers. We present the first worldwide study on the apple fruit microbiome that examines questions regarding the composition and the assembly of microbial communities on and in apple fruit. Results: Results revealed that the composition and structure of the fungal and bacterial communities associated with ‘Royal Gala’ apple fruit at harvest maturity 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 suggest a potential link between location and the type and rate of postharvest diseases that develop in each country. Although the geography, climatic conditions, and management practices may have a significant impact on the composition of fruit microbial communities, we were able to identify a 'core' microbiome that is shared in fruit across the globe. Conclusions: Results of this study provide 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.

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. info:eu-repo/semantics/acceptedVersion

En la actualidad, la normativa europea vigente ((UE) No 546/2011) exige una evaluación rigurosa de los riesgos antes de registrar un fungicida biológico como producto fitosanitario para poscosecha de fruta, y antes de su introducción en el mercado. Esta evaluación debe incluir información sobre la capacidad del agente de control biológico (ACB) para colonizar, persistir y propagarse, así como las posibles rutas de dispersión en condiciones habituales de uso. Los métodos moleculares basados en la detección de ácidos nucleicos por medio de la técnica de reacción en cadena de la polimerasa (PCR) pueden ser de gran ayuda para investigadores que trabajan en control biológico. En este trabajo, brevemente se introduce el programa de desarrollo de un ACB, y se presentan ejemplos de cómo las herramientas moleculares han sido utilizadas para generar información valiosa para el registro de un ACB efectivo contra patógenos de poscosecha de fruta.

Mechanisms that control of root system architecture are well characterised but little is known about how these processes respond to plant growth promoting rhizobacteria. We therefore studied how the presence of Pseudomonas oryzihabitans PGP01 altered wild type RSA and how these changes were modified in mutants that are defective antioxidant capacity (vtc2-1, vtc2-2, pad2-1, cad2-1 and rax1-1) or strigolactone (SL) synthesis (max3-9 and max4-1) or signalling (max2-3). The presence of P. oryzihabitans PGP01 decreased the length of primary and lateral roots but increased the number of lateral roots and lateral root density in the wild type roots but not in the SL mutants. The presence of synthetic SL, GR24 in combination with P. oryzihabitans PGP01 significantly decreased the number and length of lateral roots in the WT, max3-9 and max4-1 but not max2-3 seedlings. Lateral root density was increased in all genotypes in the presence of bacterium, but this effect was less pronounced in the ascorbate deficient vtc2-1 and vtc2-2 roots and absent from glutathione –deficient (pad2-1, cad2-1 and rax1-1) seedlings. Taken together, these results demonstrate the importance of SL-mediated signalling in root responses to growth promoting rhizobacteria, as well roles of cellular redox controls in these processes.

In the present study, two fungi Cladosporium ramotenellum strain PGP02 and Phoma spp. strain PGP03 and the bacterium Pseudomonas oryzihabitans PGP01 were isolated from Pyrus and Prunus in vitro rescued embryos, whose plantlets showed a better growth than non-contaminated cultures. Upon identification, concentrated solutions of the three microorganisms were applied to pear (Pyrus communis L.) in vitro rooted plantlets, increasing in different ways biometric parameters such as plant fresh weight (FW), stem length and root length. Then, these microorganisms were tested in embryos derived from three directed crosses between early ripening nectarine varieties (Prunus persica cv. Nectarina). In a first cross, in vitro cultured embryos were inoculated with both fungi, C. ramotenellum PGP02 and Phoma spp. PGP03, at 2 × 107 esp mL−1, and the bacterium P. oryzihabitans PGP01, at 2 × 108 CFU mL−1. In the following crosses, only the bacterium P. oryzihabitans PGP01, at 2 × 108 CFU mL−1, was employed. The effects on number of germinated embryos, development of the subsequent plants, after 24 weeks of in vitro culture, as well as their ex vitro acclimatization performance were analysed. These microorganisms had no effect on the germination efficiency of nectarine embryos. However, the presence of the bacterium P. oryzihabitans PGP01 modified root system architecture in the three crosses, increasing root volume and thickness, which in consequence enhanced the acclimatization efficiency to soil in those crosses with poor acclimation efficiencies. These results enforce a breakthrough in the use of microorganisms along the in vitro embryo rescue used in early ripening peaches and nectarines breeding programmes, and the production of plants more resistant to the stressful conditions imposed by the acclimatization to soil. info:eu-repo/semantics/acceptedVersion

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. info:eu-repo/semantics/acceptedVersion

Ampelomyces quisqualis has been reported to be a promising biocontrol agent (BCA) against powdery mildew, and it has been commercialised as AQ10 Biofungicide®. However, isolated strains showed inconsistent results when applied under practical conditions, and research to find a thoroughly effective strain is still in progress. In addition, asexual spores of the BCA are required to parasitise powdery mildew; however, producing a high amount of conidia is difficult. The present study aimed to select of an effective strain of A. quisqualis against powdery mildew on zucchini leaves and to test its conidiation in liquid media. The efficacy of several isolates from cucurbits were tested and the most effective was identified as A. quisqualis CPA-9, which reduced powdery mildew on zucchini leaves up to 61.5 % when it was applied at 2.5 × 105 conidia/mL. Conidial production of CPA-9 in liquid media was optimised, and several growing conditions were tested, such as the medium, agitation, and incubation time. Asexual reproduction was induced by modifying the aw of the media or reducing agitation. A concentration of 7.3 × 107 conidia/mL was achieved in 500-mL Erlenmeyer flasks containing 75 mL of potato dextrose broth modified with 2.5 % (w/v) of glycerol incubated at 25 °C for 11 days in the dark and without agitation. The produced conidia of CPA-9 maintained the biocontrol efficacy against P. xanthii on zucchini plants and reduced the disease incidence by up to 83 %. info:eu-repo/semantics/acceptedVersion

Highly productive agriculture depends on the application of chemical pesticides which may have negative impacts on human health and the environment. Biological control based on the use of microbial antagonists is a very promising alternative disease control method. Unfortunately, its application at a commercial scale has encountered several difficulties. In addition to financial and regulatory issues, there are two important obstacles in the development of a successful biological control agent: the difficulty of developing a formulation that preserves viability and efficacy for long storage periods and the susceptibility of the microorganism to stressful environmental conditions. In this Chapter, possible solutions are offered to overcome the issues previously described. First of all, the use of modified growth media that favors the accumulation of compatible solutes in the microbial cells can be utilized to obtain formulations with higher tolerance to unfavorable environmental conditions and to dehydration during formulation. Moreover, significant impacts on cell survival and shelf-life can obtained by the use of protectants during the dehydration processes or the use of isotonic solutions for preservation of liquid formulations. Furthermore, the use of coating forming additives mixed with the biological control agent in the treatment broth has proven to have benefits in the colonization and persistence of the microorganism on plant tissues. Finally, two innovative processes that have been developed recently are described: the use of a fluidized-bed spray-dryer to obtain dry formulations with several advantages over the more commonly used spray-drying, freeze drying and fluid bed drying systems and the use of formulations with the ability to form films on plant surfaces without the need of extra additives in the spray mix.

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/acceptedVersion

This is the peer reviewed version of the following article: Carbo, A., Torres, R., Usall, J., Marin, A., Chiralt, A. and Teixido, 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.

10 pág., 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., This work was carried out with the financial support from BIOCOMES project (EU FP7 612713).

The survival of the fungus Monilinia fructicola on fruit and inert surfaces at different temperatures (range: 0–30°C) and relative humidity (RH) (range: 60–100%) was investigated. M. fructicola conidia survived better on fruit than on inert surfaces. The viability reduction rate at 20°C and 60% RH was 1.2 and 5.8 days⁻¹ on fruit and inert surfaces, respectively. Overall, on fruit surfaces, conidia viability was reduced at high temperatures and was longer at higher RH than at lower RH; in contrast, on inert surfaces, conidia viability was longer at only low temperatures. On fruit surfaces, at 0°C and 100% RH, conidia survived up to 35 days, and at 30°C and 60% RH, conidia survived up to 7 days. However, on inert surfaces at 20°C and 30°C, conidia lost their viability after 48 and 24 h, respectively. These results suggest that M. fructicola can remain viable in cold rooms for over 30 days on fruit surfaces or over 25 days on inert surfaces. Furthermore, under the orchard conditions during the growing season, conidia may remain viable for only 2–3 days on immature fruit surfaces before conidia will be unable to penetrate the host.

BACKGROUND Bacillus amyloliquefaciens strain CPA-8 has been described as an effective biocontrol agent to control brown rot in stone fruit for both preharvest and postharvest applications. However, no information about the environmental fate and behaviour of this strain under field conditions is available. RESULTS The dispersion of the CPA-8 application was evaluated using water-sensitive papers, and complete coverage was observed on the leaves of treated trees, while

The biocontrol agent Candida sake CPA-1 has demonstrated to be effective against several diseases on fruit. However, for application of CPA-1 under field conditions, it was necessary to mix it with a food coating to improve survival under stress conditions, as well as adherence and distribution on fruit surfaces. The objective of this study was to obtain a more competitive formulation under field conditions to be applied independently of any product. To achieve this purpose, the drying process of CPA-1 by a fluidised-bed spray-drying system together with biodegradable coatings was optimised. This approach is novel for the drying system used and the formulation obtained which was able to form a film or coating on fruit surfaces. Several substances were tested as carriers and binders, and drying temperature was optimised. The addition of protective compounds was also tested to improve survival of CPA-1 during the dehydration process. Product shelf life, biocontrol efficacy on grapes against Botrytis cinerea, and the improvement of C. sake behaviour under stress conditions were tested. The optimal temperature of drying was 55 °C and two formulations that were able to develop a coating on fruit surfaces were obtained. One of the formulations was created by using a combination of native and pregelatinised potato starch; the other formulation was obtained using maltodextrin and by adding skimmed milk and sucrose as protectant compounds. The formulated products reduced the incidence and severity of B. cinerea, and CPA-1 survival rate was increased under stress conditions of temperature and humidity.

BACKGROUND Brown rot caused by Monilinia spp. is one of the most important postharvest diseases of stone fruit. The aim of this study was evaluate the relevance of the main postharvest operations of fruit; hydrocooling, cold room, water dump, sorting and cooling tunnel in the development of M. laxa on peaches and nectarines artificially infected 48, 24 or 2 hours before postharvest operations. RESULTS Commercial hydrocooling operation reduced incidence to 10 % in ‘Pp 100’ nectarine inoculated 2 and 24 hours before this operation however in ‘Fantasia’ nectarine incidence was not reduced, although lesion diameter was decreased in all studied varieties. Hydrocooling operation during 10 min and 40 mg L−1 of sodium hypochlorite reduced brown rot incidence between 50 to 77 % in nectarines inoculated 2 hours before operation, however in peaches varieties was not reduced. Water dump operation showed reduction of incidence on nectarine infected 2 hours before immersion during 30 seconds in clean water at 4 °C and 40 mg L−1 of sodium hypochlorite however in peaches varieties was not reduced. Cold room, sorting and cooling tunnel operation did not reduce brown rot incidence. CONCLUSION From all studied handling operations on stone fruits packinghouses, hydroccoling is the most relevant on the development of brown rot disease. Duration of the treatment seems to be more important than chlorine concentration. In addition, hydrocooling and water dump were less relevant on peaches than in nectarines. As a general trend, hydrocooling and water dump reduced incidence on fruit with recent infections (2 or 24 hours before operation) however when infections have been established (48 hours before operation) diseases was not reduced.

The present work focuses on the antifungal effect of volatile organic compounds (VOCs) produced by Bacillus amyloliquefaciens CPA-8 against Monilinia laxa, M. fructicola and Botrytis cinera, three postharvest fruit pathogens of sweet cherry fruit. VOCs were evaluated with a double petri dish assay against mycelial and colony growth of target pathogens. For this purpose, CPA-8 was grown on different media and cultured for 24 and 48 h at 30 °C before assays. Data showed that mycelial growth inhibition was higher when CPA-8 was grown on Tryptone Soya Agar (TSA) while no differences were generally observed when CPA-8 was cultured for either, 24 and 48 h. Moreover, no effects were observed on colony growth. The main volatile compounds emitted by CPA-8 were identified by solid-phase microextraction (SPME)-gas chromatography as 1,3 pentadiene, acetoin (3-hydroxy-2-butanone) and thiophene. Pure compounds were also tested in vitro on mycelial growth inhibition and their EC50 values against the three pathogens were estimated. Thiophene was the most effective VOC, showing more than 82% suppression of mycelial growth at the highest concentration (1.35 μL/mL headspace) and EC50 values ranging from 0.06 to 6.67 μL/mL headspace. Finally, the effectiveness of thiophene and CPA-8 VOCs was evaluated against artificially inoculated cherry fruits. Among the target pathogens, M. fructicola was clearly controlled by CPA-8 with less than 25% of rotten fruits compared to the control (65% disease incidence) and for all pathogens, less than 37.5% of CPA-8 treated decayed fruits produced spores (disease sporulation). Otherwise, pure thiophene showed no effect against any pathogen on disease incidence and disease sporulation. The results indicated that VOCs produced by B. amyloliquefaciens CPA-8 could develop an additive antifungal effect against postharvest fruit pathogens on stone fruit. This research was supported by the European project BIOCOMES FP7-612713 and by the Catalan government (Generalitat de Catalunya) for the PhD grant 2016-FI-B2 00143 (Amparo M. Gotor).

Although several studies have reported the stress-induced ethylene burst occurring in fruit in response to pathogen attack, relatively little is known about the role of this hormone in the fungal metabolism of many postharvest pathogens. To gain further knowledge, we investigated the ethylene production capacity and which ethylene biosynthetic pathways (including the 1-aminocyclopropane-1-carboxylic acid (ACC), the 2-oxoglutarate (OXO) and the 2-keto-4-methylthiobutyric acid (KMBA) pathways) were used under different in vitro conditions by Penicillium digitatum and Penicillium expansum. Both pathogens were capable of producing ethylene in vitro using different pathways but were dependant on the growing conditions. For instance, both the KMBA and the OXO pathways seemed to be responsible for ethylene biosynthesis in P. digitatum when grown in more stressful conditions (limited surface and nutrient availability as in PDA tubes), while only the KMBA pathway seemed to be activated under other culture conditions (i.e. on PDA plates) or when the fungal mycelium was placed within aqueous solutions containing ethylene precursors. In contrast, the KMBA appeared to be the exclusive pathway responsible for ethylene production by P. expansum yet only when the fungal mycelium was in contact with aqueous solutions containing ethylene precursors. In both pathogens, ethylene production seemed to be synchronized with certain stages of fungal growth rather than conidiation, suggesting a putative role of this hormone in fungal metabolism.

The biocontrol agent Candida sake CPA-1 is effective against several diseases. Consequently, the optimisation of a dry formulation of C. sake to improve its shelf life and manipulability is essential for increasing its potential with respect to future commercial applications. The present study aimed to optimise the conditions for making a dry formulation of C. sake using a fluidised bed drying system and then to determine the shelf life of the optimised formulation and its efficacy against Penicillium expansum on apples.; Results: The optimal conditions for the drying process were found to be 40 °C for 45 min and the use of potato starch as the carrier significantly enhanced the viability. However, none of the protective compounds tested increased the viability of the dried cells. A temperature of 25 °C for 10 min in phosphate buffer was considered as the optimum condition to recover the dried formulations. The dried formulations should be stored at 4 °C and air-packaged; moreover, shelf life assays indicated good results after 12 months of storage. The formulated products maintained their biocontrol efficacy.; Conclusion: A fluidised bed drying system is a suitable process for dehydrating C. sake cells; moreover, the C. sake formulation is easy to pack, store and transport, and is a cost-effective process. © 2017 Society of Chemical Industry.; © 2017 Society of Chemical Industry.

Biological control with microorganisms is regarded as a promising control strategy against Botrytis bunch rot (BBR, grey mould) on grapes. Candida sake CPA-1 is a yeast that has previously shown a high efficacy against BBR in small-scale field trials. The present work aims to confirm this efficacy in commercial conditions and to evaluate the compatibility of C. sake with phytosanitary products that are commonly used in viticulture. The large-scale spray programmes (two to three applications in the growing season) were carried out in three field sites in Catalonia, North-East Spain, with different vineyard management styles. The overall reductions were in the ranges of 35 ± 5.7 to 64 ± 3.7% incidence, and 39 ± 17.5 to 85 ± 3.9% severity, compared to the control. Only four out of 39 phytosanitary products, tested in liquid suspension with C. sake, were incompatible with the yeast. The results demonstrated the potential of C. sake to become a reliable solution as a biocontrol product to be integrated in the control of BBR within the future.

Monilinia spp. is the main pathogen responsible for postharvest losses of stone fruit. Several studies have examined the conditions for Monilinia spp. infection in the field, but very limited information is available about postharvest. Storing fruit for 1 day in the cold room or dumping fruit in a water tank are the most common handling operations during the postharvest of fruit. Then, the aim of this study was to investigate the risk of Monilinia fructicola infection for two peach and one nectarine cultivars during cold storage and water dump operations. A new methodology was performed using a dry inoculum of M. fructicola. A set of fruit was used as control to demonstrated that at 20 °C 60% relative humidity (RH) was not able to infect fruit, however, the disease was developed when was already infected. In addition, M. fructicola was able to infect and develop disease in fruit at 20 °C 100 RH. The storage of fruit with the presence of M. fructicola conidia on their surface for up to 30 days at 0 °C 100% RH or 4 °C 100% RH did not suppose an important risk of infection since only 3.3 and 3.8%, respectively of fruit were already infected. Overall, all treatments tested with the water dump operation gave optimal conditions to M. fructicola to infect fruit when it was superficially dry inoculated or it was immersed with water contaminated with conidia, increasing the need for water disinfection. In addition, when fruit was immersed in water free from M. fructicola conidia, the postharvest operation gave optimal conditions to develop infections already produced. info:eu-repo/semantics/acceptedVersion

[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., This study was supported by national projects AGL2014‐55287‐C02‐02‐R and RTA2015‐00050‐00‐00 from the Ministry of Economy, Industry and Competitiveness (MINECO), by a PhD grant 2017FI_B1_00153 (Baró‐Montel, N.) from the Catalan Government (Generalitat de Catalunya) and by funding received from the CERCA Programme/Generalitat de Catalunya and the Severo Ochoa Program for Centres of Excellence in R&D 2016‐2019 (SEV‐2015‐0533).

Agrochemical companies are increasing their interest in the production of biopesticides based on biocontrol agents (BCAs), mainly due to the need to reduce the synthetic fungicide application and the consequent benefits for the environment and for human health. The efficacy of the BCA Candida sake CPA-1 has been demonstrated against the main postharvest diseases on pome fruit, and B. cinerea and sour rot in grapes control on field. Recently, two effective fluidised-bed spray-dried formulations based on C. sake CPA-1 and biodegradable compounds were described as an effective biocontrol product. However, the maintenance of the effectiveness and survival of these formulations during a long shelf life is a crucial aspect to the success of its marketing. For this reason, the aim of this study was to select the best packaging and storage conditions to maintain a long shelf life of two fluidised-bed spray-dried C. sake CPA-1 formulations. Viability and aw (water activity) of CPA-1 formulations stored in different packages (bottles and bags), temperatures (− 20 °C and 4 °C) and atmospheric conditions (vacuum and air) were tested for 22 months. Both formulations maintained their initial viability (around 4 × 109 CFU ml−1) when they were stored at − 20 °C regardless of the packages or atmospheric conditions. In contrast, the viability of CPA-1 formulations stored at 4 °C differed between bags and bottles. Both formulations could be stored for at least 21 months at − 20 °C, and only one formulation could achieve a one-year shelf life stored in bottles at 4 °C. C. sake CPA-1 formulations stored for 12 months at the most suitable conditions significantly reduced B. cinerea on grapes by 25 to 56%. info:eu-repo/semantics/acceptedVersion