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BACKGROUND: Growing concerns about sustainability have driven the search for eco-friendly pest management solutions. Combining natural and synthetic compounds within controlled release systems is a promising strategy. This study investigated the co-encapsulation of the natural compound citral (Cit) and the synthetic antifungal cyproconazole (CPZ) using two water-based nanocarriers: solid lipid nanoparticles (SLNs) and chitosan nanoparticles (CSNPs). RESULTS: Both CSNPs and SLNs loaded with Cit + CPZ displayed superior antifungal activity against Botrytis cinerea compared to free compounds. Notably, CSNPs with a 2:1 Cit:CPZ ratio exhibited the highest efficacy, achieving a minimum inhibitory concentration (MIC100) of < 1.56 µg mL-1, lower than the 12.5 µg mL-1 of non-encapsulated compounds. This formulation significantly reduced the required synthetic CPZ while maintaining efficacy, highlighting its potential for environmentally friendly pest control. CONCLUSION: The successful co-encapsulation of Cit + CPZ within CSNPs, particularly at a 2:1 ratio, demonstrates a promising approach for developing effective and sustainable antifungal formulations against B. cinerea. © 2024 Society of Chemical Industry.

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The present study investigates the interactions between eight glucosinolate hydrolysis products (GHPs) sourced from broccoli by-products and the detoxifying enzymes of Botrytis cinerea, namely eburicol 14-alpha-demethylase (CYP51) and glutathione-S-transferase (GST), through in silico analysis. Additionally, in vitro assays were conducted to explore the impact of these compounds on fungal growth. Our findings reveal that GHPs exhibit greater efficacy in inhibiting conidia germination compared to mycelium growth. Furthermore, the results demonstrate the antifungal activity of glucosinolate hydrolysis products derived from various parts of the broccoli plant, including inflorescences, leaves, and stems, against B. cinerea. Importantly, the results suggest that these hydrolysis products interact with the detoxifying enzymes of the fungus, potentially contributing to their antifungal properties. Extracts rich in GHPs, particularly iberin and indole-GHPs, derived from broccoli by-products emerge as promising candidates for biofungicidal applications, offering a sustainable and novel approach to plant protection by harnessing bioactive compounds from agricultural residues.

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Botrytis cinerea is the causal agent of gray mold, which affects a wide variety of plant species. Chemical agents have been used to prevent the disease caused by this pathogenic fungus. However, their toxicity and reduced efficacy have encouraged the development of new biological control alternatives. Recent studies have shown that bacteria isolated from amphibian skin display antifungal activity against plant pathogens. However, the mechanisms by which these bacteria act to reduce the effects of B. cinerea are still unclear. From a diverse collection of amphibian skin bacteria, three proved effective in inhibiting the development of B. cinerea under in vitro conditions. Additionally, the individual application of each bacterium on the model plant Arabidopsis thaliana, Solanum lycopersicum and post-harvest blueberries significantly reduced the disease caused by B. cinerea. To understand the effect of bacteria on the host plant, we analyzed the transcriptomic profile of A. thaliana in the presence of the bacterium C32I and the fungus B. cinerea, revealing transcriptional regulation of defense-related hormonal pathways. Our study shows that bacteria from the amphibian skin can counteract the activity of B. cinerea by regulating the plant transcriptional responses.

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Fungi are a problem for viticulture as they can lead to deterioration of grapes and mycotoxins production. Despite the widespread use of synthetic fungicides to control fungi, their impact on the agricultural ecosystem and human health demand safer and eco-friendly alternatives. This study aimed to produce, characterize and assess the antifungal activity of carvacrol loaded in nanocapsules of Eudragit® and chia mucilage as strategy for controlling Botrytis cinerea, Aspergillus flavus, Aspergillus carbonarius, and Aspergillus niger. Eudragit® and chia mucilage were suitable wall materials, as both favored the encapsulation of carvacrol into nanometric diameter particles. Fourier Transform Infrared Spectroscopy (FTIR) analysis suggested a successful incorporation of carvacrol into both nanocapsules, which was confirmed by presenting a good encapsulation efficiency and loading capacity. Thermogravimetric Analysis (TGA) and Differential Scanning Calorimetry (DSC) analyses revealed adequate thermal resistance. All fungi were sensible to carvacrol treatments and B. cinerea was the most sensitive compared to the Aspergillus species. Lower concentrations of encapsulated carvacrol than the unencapsulated form were required to inhibit fungi in the in vitro and grape assays. Additionally, lower levels of carvacrol (unencapsulated or encapsulated) were used to inhibit fungal growth and ochratoxin synthesis on undamaged grapes in comparison to those superficially damaged, highlighting the importance of management practices designed to preserve berry integrity during cultivation, storage or commercialization. When sublethal doses of carvacrol were used, the growth of A. niger and A. carbonarius was suppressed by at least 45 %, and ochratoxins were not found. The nanoencapsulation of carvacrol using Eudragit® and chia mucilage has proven to be an alternative to mitigate the problems with fungi and mycotoxins faced by the grape and wine sector.

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The dragon fruit is native of Mexico, and Puebla is the third-largest producing state (SIAP 2023). In June 2023, field sampling was conducted in El Paraíso, Atlixco (18° 49' 5.275" N, 98° 26' 52.353" W), Puebla, Mexico. The mean temperature and relative humidity were 20 °C and 75% for seven consecutive days. Dragon fruits cv. 'Delight' close to harvest with gray mold symptoms were found in a commercial area of 2 ha, with an incidence of 35 to 40% and an estimated severity of 75% on infected fruit. The symptoms included necrosis at the apex, which later spread throughout the fruit, along with a soft, black rot covered in abundant mycelium and sporulation. The fungus was isolated from 40 symptomatic fruits by disinfesting pieces of necrotic tissue with 3% NaClO for one minute, rinsing with sterile distilled water (SDW), plating on Petri dishes with potato dextrose agar, and incubating at 25 °C in the dark. One isolate was obtained from each diseased fruit by the hyphal-tip method. The colonies were initially white with a growth rate of 1.15-1.32 cm per day and turned gray after 10 days; the mycelium was dense and aerial. Spherical and irregular sclerotia were formed, measuring 0.9-1.4 × 0.6-1.1 mm (n = 100). Each Petri dish produced 56-278 sclerotia (n = 40) after 11 days; these were initially white and gradually turned dark brown. Brown to olive conidiophores were straight, septate, and branched, measuring 1075-1520 × 10-21 µm, with elliptical hyaline to light brown conidia of 6.6-11.5 × 5-8.1 µm (n=100). The isolates were tentatively identified as Botrytis cinerea based on morphological characteristics (Ellis 1971). Two representative isolates were chosen for molecular identification and genomic DNA was extracted by the CTAB protocol. The ITS region and the heat shock protein (HSP60), RNA polymerase binding II (RPB2) and glyceraldehyde 3-phosphate dehydrogenase (G3PDH) genes were sequenced (White et al. 1990; Staats et al. 2005). The sequences of a representative isolate (BcPh5) were deposited in GenBank (ITS-OR582337; HSP60-OR636622; RPB2-OR636623; and G3PDH-OR636621). BLAST analysis of the partial sequences of ITS (479 bp), HSP60 (1006 bp), RPB2 (1126 bp), and G3PDH (907 bp) showed 100% similarity to B. cinerea isolates (GenBank: KM840848, MH796663, MK919495, MF480679). Phylogenetic analysis confirmed that BcPh5 clustered with B. cinerea strains. Pathogenicity was confirmed by inoculating the non-wounded surface of 20 detached dragon fruits cv. 'Delight' using the BcPh5 isolate by depositing 20 µl of a 105 conidia/ml suspension with a sterile syringe. The fruits were placed on the rim of a plastic container and inserted in a moisture box with 2 cm of water at the bottom. The box was covered with a plastic sheet to maintain humidity. Control fruits were inoculated with SDW. The inoculated fruits became covered with abundant white to gray mycelium, and soft rot developed within eight days, while no symptoms were observed on the controls. The fungus was re-isolated from the inoculated fruits as described above, fulfilling Koch's postulates. The pathogenicity tests were repeated three times. Gray mold caused by B. cinerea was also recently reported in Mexico on pomegranate (Hernández et al. 2023) and rose apple (Isodoro et al. 2023). As far as we know, this is the first report of B. cinerea causing gray mold on dragon fruit in Mexico. This research is essential for designing integrated management strategies against gray mold on dragon fruits.

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The main phytosanitary problem for table grape production in Chile is gray mold caused by the fungus Botrytis cinerea. To manage this issue, the primary method utilized is chemical control. Fludioxonil, a phenylpyrrole, is highly effective in controlling B. cinerea and other plant pathogens. Consistently, there have been no field reports of reduced efficacy of fludioxonil; however, subpopulations with reduced sensitivity to fludioxonil are on the rise globally, as per increasing reports. Our study involved a large-scale evaluation of B. cinerea's sensitivity to fludioxonil in the Central Valley of Chile's primary table grape production area during the growing seasons from 2015 to 2018. Out of 2,207 isolates, only 1.04% of the isolates (n = 23) exceeded the sensitivity threshold value of 1 µg/ml. Remarkably, 95.7% are concentrated in a geographic region (Valparaíso Region). Isolates with reduced sensitivity to fludioxonil showed growth comparable with sensitive isolates and even more robust growth under nutritional deficit, temperature, or osmotic stress, suggesting greater environmental adaptation. When table grape detached berries were stored at 0°C, isolates less sensitive to fludioxonil caused larger lesions than sensitive isolates (2.82 mm compared with 1.48 mm). However, the lesions generated by both types of isolates were equivalent at room temperature. This study found no cross-resistance between fludioxonil and fenhexamid, an essential fungicide integrated with fludioxonil in Chilean B. cinerea control programs. All the Chilean isolates with reduced sensitivity to fludioxonil were controlled by the fludioxonil/cyprodinil mixture, a commonly employed form of fludioxonil. The cyprodinil sensitivity in the isolates with reduced sensitivity to fludioxonil explains their low field frequency despite their null fitness penalties. However, the emergence of fludioxonil-resistant isolates inside the Chilean B. cinerea population demands a comprehensive analysis of their genetic bases, accompanied by monitoring tools that allow the permanence of field fludioxonil efficacy.

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In vitro antimicrobial activity of nano-ZnO-loaded nanoliposomes at different levels of lecithin:nano-ZnO ratio (5:1, 15:1, and 25:1 w/w) against Aspergillus niger (IBRC-M 30095) and Botrytis cinerea (IBRC-M 30162) was evaluated. Nanoliposome formulations containing nano-ZnO were fabricated through thin-layer hydration sonication and heat methods. The minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of nano-ZnO-loaded nanoliposomes and free nano-ZnO against Aspergillus niger and Botrytis cinerea were determined. The time-kill experiments were performed for each isolate. Results showed that the encapsulation of nano-ZnO in nanoliposome systems significantly enhanced their antimicrobial activities by improving the penetration of ZnO nanoparticles the fungi cell membrane. In vitro antifungal activity of nano-ZnO-loaded nanoliposomes against Aspergillus niger and Botrytis cinerea was increased in thin-layer hydration sonication method compared with the heat method. The log phase for Aspergillus niger and Botrytis cinerea was around 70 h. Adding nano-ZnO-loaded nanoliposomes to the culture medium shortened the log phase for both Aspergillus niger and Botrytis cinerea. The highest antimicrobial activity of nanoliposomes was achieved using nanoliposomes containing the lecithin:nano-ZnO ratio of 25:1 (w/w) as compared to all samples. However, the length of the log phase growth cultures exposed to the nanoliposome formulations prepared by thin-layer hydration sonication method with the lecithin:nano-ZnO ratio of 25:1 (w/w) at MIC and MFC values was 60 and 40 h for both Aspergillus niger and Botrytis cinerea, respectively.

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A biodegradable polymer packaging system for 'Benitaka' table grapes (Vitis vinifera L.) was developed to inhibit the development of gray mold during refrigerated storage. The system consisted of packages and sachets containing Na2S2O5 to release sulfur dioxide (SO2), both produced with biodegradable films of starch, glycerol, and poly (adipate co-butylene terephthalate) (PBAT) produced via blown extrusion. The films were characterized in terms of thickness, density, mass loss in water, water vapor permeability, sorption isotherms, and mechanical properties. The table grapes were packed with biodegradable plastic bags containing SO2-releasing sachets inside. The experimental design was completely randomized, with four repetitions and five treatments: (a) control, without sachet containing Na2S2O5 and SiO2; (b) 2 g of Na2S2O5 + 2 g of SiO2; (c) 4 g of Na2S2O5 + 1 g of SiO2; (d) 4 g of Na2S2O5 + 2 g of SiO2; and (e) 4 g of Na2S2O5 + 4 g of SiO2. The bunches were stored in a refrigerated chamber at 1 ± 1 °C and relative humidity above 90%. The treatments were evaluated 30 and 45 days after the beginning of refrigerated storage and 3 days at room temperature. The grapes were evaluated based on the incidence of gray mold, mass loss, stem browning, shattered berries, and berry bleaching. The data were subjected to the analysis of variance, and the means were compared using Tukey's test at 5%. The biodegradable films had good processability during the production via blown extrusion, with good physical properties to be used in the packaging of grapes and the production of SO2-releasing sachets. The biodegradable polymer packaging system (biodegradable plastic bags + SO2-releasing sachets) inhibited the development of gray mold on 'Benitaka' table grapes for 45 days at 1 °C, preserving their quality, with low mass loss, few shattered berries, and rachis freshness.

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Phospholipase C (PLC) plays a key role in lipid signaling during plant development and stress responses. PLC activation is one of the earliest responses during pathogen perception. Arabidopsis thaliana contains seven PLC encoding genes (AtPLC1 to AtPLC7) and two pseudogenes (AtPLC8 and AtPLC9), being AtPLC2 the most abundant isoform with constitutive expression in all plant organs. PLC has been linked to plant defense signaling, in particular to the production of reactive oxygen species (ROS). Previously, we demonstrated that AtPLC2 is involved in ROS production via the NADPH oxidase isoforms RBOHD activation during stomata plant immunity. Here we studied the role of AtPLC2 on plant resistance against the necrotrophic fungus Botrytis cinerea, a broad host-range and serious agricultural pathogen. We show that the AtPLC2-silenced (amiR PLC2) or null mutant (plc2-1) plants developed smaller B. cinerea lesions. Moreover, plc2-1 showed less ROS production and an intensified SA-dependent signaling upon infection, indicating that B. cinerea uses AtPLC2-triggered responses for a successful proliferation. Therefore, AtPLC2 is a susceptibility (S) gene that facilitates B. cinerea infection and proliferation.

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Certain metals play key roles in infection by the gray mold fungus, Botrytis cinerea. Among them, copper and iron are necessary for redox and catalytic activity of enzymes and metalloproteins, but at high concentrations they are toxic. Understanding the mechanism requires more cell characterization studies for developing new, targeted metal-based fungicides to control fungal diseases on food crops. This study aims to characterize the inhibitory effect of copper and iron on B. cinerea by evaluating mycelial growth, sensitivity to cell wall perturbing agents (congo red and calcofluor white), membrane integrity, adhesion, conidial germination, and virulence. Tests of copper over the range of 2 to 8 mM and iron at 2 to 20 mM revealed that the concentration capable of reducing mycelial growth by 50% (IC50) was 2.87 mM and 9.08 mM for copper and iron, respectively. When mixed at equimolar amounts there was a significant inhibitory effect mostly attributable to copper. The effect of Cu50, Fe50, and Cu50-Fe50 was also studied on the mycelial growth of three wild B. cinerea strains, which were more sensitive to metallic inhibitors. A significant inhibition of conidial germination was correlated with adhesion capacity, indicating potential usefulness in controlling disease at early stages of crop growth. Comparisons of the effects of disruptive agents on the cell wall showed that Cu, Fe, and Cu-Fe did not exert their antifungal effect on the cell wall of B. cinerea. However, a relevant effect was observed on plasma membrane integrity. The pathogenicity test confirmed that virulence was correlated with the individual presence of Cu and Fe. Our results represent an important contribution that could be used to formulate and test metal-based fungicides targeted at early prevention or control of B. cinerea.

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The BcWCL1 protein is a blue-light photoreceptor from the fungus Botrytis cinerea. This protein has a central role in B. cinerea circadian regulation and is an ortholog to WC-1 from Neurospora crassa. The BcWCL1 and WC-1 proteins have similar protein domains, including a LOV (Light Oxygen Voltage) domain for light sensing, two PAS (Per Arnt Sim) domains for protein-protein interaction, and a DNA binding domain from the GATA family. Recently, the blue-light response of BcWCL1 was demonstrated in a version without PAS domains (BcWCL1PAS∆). Here, we demonstrated that BcWCL1PAS∆ is capable of self-dimerization through its N-terminal region upon blue-light stimulation. Interestingly, we observed that BcWCL1PAS∆ enables transcriptional activation as a single component in yeast. By using chimeric transcription factors and the luciferase reporter gene, we assessed the transcriptional activity of different fragments of the N-terminal and C-terminal regions of BcWCL1PAS∆, identifying a functional transcriptional activation domain (AD) in the N-terminal region that belongs to the 9aaTAD family. Finally, we determined that the transcriptional activation levels of BcWCL1PAS∆ AD are comparable to those obtained with commonly used ADs in eukaryotic cells (Gal4 and p65). In conclusion, the BcWCL1PAS∆ protein self-dimerized and activated transcription in a blue-light-dependent fashion, opening future applications of this photoreceptor in yeast optogenetics.

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The postharvest disease popularly known as gray mold is considered one of the most limiting factors strawberry fruit production. The most effective way to control this disease is still the use of chemical fungicides. However, other alternative sources of control are being explored. Among these, psychrophilic yeasts adapted to extreme conditions, such as those found in the Antarctic region, may have great potential for use as biocontrol agents. Thus, the present study aimed to select psychrotolerant yeasts obtained from Antarctic region and to evaluate their potential for biocontrol under gray mold, caused by Botrytis cinerea in strawberries stored at low temperature. For this, 20 potential antagonist yeasts were evaluated in vitro (thermotolerance and enzymatic) assays. Debaryomyces hansenii, Rhodotorula mucilaginosa and Dioszegia hungarica were selected for growing in strawberry juice. However, only D. hansenii was selected for in vivo studies and showed a reduction in the incidence of gray mold by 82% for the tests performed on injury and 86% for the tests on non-injured fruits treated by immersion bath. Thus, demonstrating that the selection of this cold-adapted Antarctic yeast can be a promising strategy as a biocontrol agent used to curb the development of gray mold in strawberry fruits.

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During plant interaction with beneficial microorganisms, fungi secrete a battery of elicitors that trigger plant defenses against pathogenic microorganisms. Among the elicitor molecules secreted by Trichoderma are cerato-platanin proteins, such as EPL1, from Trichoderma atroviride. In this study, Arabidopsis thaliana plants that express the TaEPL1 gene were challenged with phytopathogens to evaluate whether expression of EPL1 confers increased resistance to the bacterial pathogen Pseudomonas syringae and the necrotrophic fungus Botrytis cinerea. Infection assays showed that Arabidopsis EPL1-2, EPL1-3, EPL1-4 expressing lines were more resistant to both pathogens in comparison to WT plants. After Pseudomonas syringae infection, there were reduced disease symptoms (e.g., small chlorotic spots) and low bacterial titers in the three 35S::TaEPL1 expression lines. Similarly; 35S::TaEPL1 expression lines were more resistant to Botrytis cinerea infection, showing smaller lesion size in comparison to WT. Interestingly, an increase in ROS levels was detected in 35S::TaEPL1 expression lines when compared to WT. A higher expression of SA- and JA-response genes occurred in the 35S::TaEPL1 lines, which could explain the resistance of these EPL1 expression lines to both pathogens. We propose that EPL1 is an excellent elicitor, which can be used to generate crops with improved resistance to broad-spectrum diseases.

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Botrytis cinerea is a necrotrophic fungus characterized mainly by its wide host range of infected plants. The deletion of the white-collar-1 gene (bcwcl1), which encodes for a blue-light receptor/transcription factor, causes a decrease in virulence, particularly when assays are conducted in the presence of light or photocycles. However, despite ample characterization, the extent of the light-modulated transcriptional responses regulated by BcWCL1 remains unknown. In this study, pathogen and pathogen:host RNA-seq analyses, conducted during non-infective in vitro plate growth and when infecting Arabidopsis thaliana leaves, respectively, informed on the global gene expression patterns after a 60 min light pulse on the wild-type B05.10 or ∆bcwcl1 B. cinerea strains. The results revealed a complex fungal photobiology, where the mutant did not react to the light pulse during its interaction with the plant. Indeed, when infecting Arabidopsis, no photoreceptor-encoding genes were upregulated upon the light pulse in the ∆bcwcl1 mutant. Differentially expressed genes (DEGs) in B. cinerea under non-infecting conditions were predominantly related to decreased energy production in response to the light pulse. In contrast, DEGs during infection significantly differ in the B05.10 strain and the ∆bcwcl1 mutant. Upon illumination at 24 h post-infection in planta, a decrease in the B. cinerea virulence-associated transcripts was observed. Accordingly, after a light pulse, biological functions associated with plant defense appear enriched among light-repressed genes in fungus-infected plants. Taken together, our results show the main transcriptomic differences between wild-type B. cinerea B05.10 and ∆bcwcl1 after a 60 min light pulse when growing saprophytically on a Petri dish and necrotrophically over A. thaliana.

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MAIN CONCLUSION: CRISPR/Cas9-mediated Phospholipase C2 knock-out tomato plants are more resistant to Botrytis cinerea than wild-type plants, with less ROS and an increase and reduction of (JA) and (SA)-response marker genes, respectively. Genome-editing technologies allow non-transgenic site-specific mutagenesis of crops, offering a viable alternative to traditional breeding methods. In this study we used CRISPR/Cas9 to inactivate the tomato Phospholipase C2 gene (SlPLC2). Plant PLC activation is one of the earliest responses triggered by different pathogens regulating plant responses that, depending on the plant-pathogen interaction, result in plant resistance or susceptibility. The tomato (Solanum lycopersicum) PLC gene family has six members, named from SlPLC1 to SlPLC6. We previously showed that SlPLC2 transcript levels increased upon xylanase infiltration (fungal elicitor) and that SlPLC2 participates in plant susceptibility to Botrytis cinerea. An efficient strategy to control diseases caused by pathogens is to disable susceptibility genes that facilitate infection. We obtained tomato SlPLC2-knock-out lines with decreased ROS production upon B. cinerea challenge. Since this fungus requires ROS-induced cell death to proliferate, SlPLC2-knock-out plants showed an enhanced resistance with smaller necrotic areas and reduced pathogen proliferation. Thus, we obtained SlPLC2 loss-of-function tomato lines more resistant to B. cinerea by means of CRISPR/Cas9 genome editing technology.

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Botrytis cinerea is a phytopathogenic fungus that causes serious damage to the agricultural industry by infecting various important crops. 2-allylphenol has been used in China as a fungicide for more than a decade, and it has been shown that is a respiration inhibitor. A series of derivatives of 2-allylphenol were synthesized and their activity against B. cinerea was evaluated by measuring mycelial growth inhibition. Results indicate that small changes in the chemical structure or the addition of substituent groups in the aromatic ring induce important variations in activity. For example, changing the hydroxyl group by methoxy or acetyl groups produces dramatic increases in mycelial growth inhibition, i.e., the IC50 value of 2-allylphenol decreases from 68 to 2 and 1 µg mL-1. In addition, it was found that the most active derivatives induce the inhibition of Bcaox expression in the early stages of B. cinerea conidia germination. This gene is associated with the activation of the alternative oxidase enzyme (AOX), which allows fungus respiration to continue in the presence of respiratory inhibitors. Thus, it seems that 2-allylphenol derivatives can inhibit the normal and alternative respiratory pathway of B. cinerea. Therefore, we believe that these compounds are a very attractive platform for the development of antifungal agents against B. cinerea.

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For comprehensive gene expression analyses of the phytopathogenic fungus Botrytis cinerea, which infects a number of plant taxa and is a cause of substantial agricultural losses worldwide, we developed BEB, a web-based B. cinerea gene Expression Browser. This computationally inexpensive web-based application and its associated database contain manually curated RNA-Seq data for B. cinerea. BEB enables expression analyses of genes of interest under different culture conditions by providing publication-ready heatmaps depicting transcript levels, without requiring advanced computational skills. BEB also provides details of each experiment and user-defined gene expression clustering and visualization options. If needed, tables of gene expression values can be downloaded for further exploration, including, for instance, the determination of differentially expressed genes. The BEB implementation is based on open-source computational technologies that can be deployed for other organisms. In this case, the new implementation will be limited only by the number of transcriptomic experiments that are incorporated into the platform. To demonstrate the usability and value of BEB, we analyzed gene expression patterns across different conditions, with a focus on secondary metabolite gene clusters, chromosome-wide gene expression, previously described virulence factors, and reference genes, providing the first comprehensive expression overview of these groups of genes in this relevant fungal phytopathogen. We expect this tool to be broadly useful in B. cinerea research, providing a basis for comparative transcriptomics and candidate gene identification for functional assays.

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The study evaluated the conservation of strawberries treated with crude plant extracts (barbatimão, sibipiruna, guarana, and catuaba) against fungal deterioration and physicochemical characteristics. MIC of 0.125; 0.0156; 0.25 and 0.0312 g/mL were found for barbatimão, sibipiruna, guaraná and catuaba, respectively, against B. cinerea. Treated samples showed no fungal deterioration during 11 days. Analyzes of weight loss, soluble solids, titratable acidity, and pH variation were performed. Sibipiruna showed lower values ​​of mass loss, and the greatest occurred for the catuaba extract. Barbatimão did not change soluble solids and stood out with catuaba in the color parameters L and a*. Small changes in pH were observed with time. Soluble solids maintained values ​​between 6.47 oBrix and 9.90 oBrix for catuaba and sibipiruna extracts at zero and six days. Principal component analysis did not show a strong correlation between the variables. The extracts become alternatives for strawberry conservation, increasing conservation and maintaining physicochemical characteristics.

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Botrytis cinerea, the causal agent of grey mold disease, is one of the most destructive pathogens of strawberry crops, both in vegetative development and postharvest. The control of this pathogen is complex due to its aggressiveness and ability to attack and infect various plant tissues and is mainly based on chemical control; however, the incorrect use of pesticides, mainly due to overdosing, causes the presence of traces of these agrochemicals in the fruits, as well as the selection of pathogen resistance to fungicides, making it a risk to human health and the environment. The objective of the study was to use biological regulation strategies, with the application of microbial consortia made up of mycorrhizal fungi, antagonistic bacteria and Trichoderma harzianum, as an alternative for the management of grey mold in strawberry crops (Monterey variety) under field conditions. Treatments T4 (mycorrhizal fungi), T8 (mycorrhizal fungi, antagonistic bacteria and T. harzianum) and T2 (T. harzianum) presented the lowest incidence of the pathogen with 2.6, 3.1 and 3.6 %, respectively, compared to control plants with 16.6%. The influence of all biological treatments on the regulation of B. cinerea was greater than the control.

Botrytis cinerea, el agente causal de la enfermedad del moho gris, es uno de los patógenos más destructivos del cultivo de fresa, tanto en el desarrollo vegetativo como en poscosecha. El control de este patógeno es complejo, debido a su agresividad y capacidad de atacar e infectar diversos tejidos de la planta y se basa, principalmente, en el control químico; sin embargo, el uso incorrecto de plaguicidas, principalmente por sobredosificación, provoca la presencia de trazas de estos agroquímicos en los frutos, así como la selección de resistencia del patógeno a los fungicidas, convirtiéndolo en un riesgo para la salud humana y el ambiente. El objetivo del estudio fue utilizar estrategias de regulación biológica, con la aplicación de consorcios microbianos, conformados por hongos micorrícicos, bacterias antagonistas y Trichoderma harzianum, como alternativa para el manejo del moho gris, en cultivos de fresa (variedad Monterey), en condiciones de campo. Los tratamientos T4 (hongos micorrízicos), T8 (hongos micorrízicos, bacterias antagonistas y T. harzianum) y T2 (T. harzianum) presentaron la menor incidencia del patógeno, con 2,6, 3,1 y 3,6 %, respectivamente, en comparación con las plantas control, con 16,6 %. La influencia de todos los tratamientos biológicos en la regulación de B. cinerea fue mayor respecto al control.

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Circadian clocks are important for an individual's fitness, and recent studies have underlined their role in the outcome of biological interactions. However, the relevance of circadian clocks in fungal-fungal interactions remains largely unexplored. We sought to characterize a functional clock in the biocontrol agent Trichoderma atroviride to assess its importance in the mycoparasitic interaction against the phytopathogen Botrytis cinerea. Thus, we confirmed the existence of circadian rhythms in T. atroviride, which are temperature-compensated and modulated by environmental cues such as light and temperature. Nevertheless, the presence of such molecular rhythms appears to be highly dependent on the nutritional composition of the media. Complementation of a clock null (Δfrq) Neurospora crassa strain with the T. atroviride-negative clock component (tafrq) restored core clock function, with the same period observed in the latter fungus, confirming the role of tafrq as a bona fide core clock component. Confrontation assays between wild-type and clock mutant strains of T. atroviride and B. cinerea, in constant light or darkness, revealed an inhibitory effect of light on T. atroviride's mycoparasitic capabilities. Interestingly, when confrontation assays were performed under light/dark cycles, T. atroviride's overgrowth capacity was enhanced when inoculations were at dawn compared to dusk. Deleting the core clock-negative element FRQ in B. cinerea, but not in T. atroviride, was vital for the daily differential phenotype, suggesting that the B. cinerea clock has a more significant influence on the result of this interaction. Additionally, we observed that T. atroviride clock components largely modulate development and secondary metabolism in this fungus, including the rhythmic production of distinct volatile organic compounds (VOCs). Thus, this study provides evidence on how clock components impact diverse aspects of T. atroviride lifestyle and how daily changes modulate fungal interactions and dynamics.

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