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Objectives To screen high active volatile organic compounds (VOCs)-producing Trichoderma isolates against strawberry gray mold caused by Botrytis cinerea, and to explore their antagonistic mode of action against the pathogen. VOCs produced by nine Trichoderma isolates (Trichoderma atroviride T1 and T3; Trichoderma harzianum T2, T4 and T5; T6, T7, T8 and T9 identified as Trichoderma asperellum in this work) significantly inhibited the mycelial growth (13.9-63.0% reduction) and conidial germination (17.6-96.3% reduction) of B. cinerea, the highest inhibition percentage belonged to VOCs of T7; in a closed space, VOCs of T7 shared 76.9% and 100% biocontrol efficacy against gray mold on strawberry fruits and detached leaves, respectively, prolonged the fruit shelf-life by 3 days in presence of B. cinerea, completely protected the leaves from B. cinerea infecting; volatile metabolites of T7 damaged the cell membrane permeability and integrity of B. cinerea, thereby inhibiting the mycelial growth and conidial germination. Gas chromatography-mass spectrometry (GC-MS) analysis revealed the VOCs contain 23 potential compounds, and the majority of these compounds were categorised as alkenes, alcohols, and esters, including PEA and 6PP, which have been reported as substances produced by Trichoderma spp. T. asperellum T7 showed high biofumigant activity against mycelial growth especially conidial germination of B. cinerea and thus protected strawberry fruits and leaves from gray mold, which acted by damaging the pathogen's plasma membrane and resulting in cytoplasm leakage, was a potential biofumigant for controlling pre- and post-harvest strawberry gray mold., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Infection of grapevines with the grey mold pathogen Botrytis cinerea results in severe problems for winemakers worldwide. Browning of wine is caused by the laccase-mediated oxidation of polyphenols. In the last decades, Botrytis management has become increasingly difficult due to the rising number of resistances and the genetic variety of Botrytis strains. During the search for sustainable fungicides, polyphenols showed great potential to inhibit fungal growth. The present study revealed two important aspects regarding the effects of grape-specific polyphenols and their polymerized oxidation products on Botrytis wild strains. On the one hand, laccase-mediated oxidized polyphenols, which resemble the products found in infected grapes, showed the same potential for inhibition of growth and laccase activity, but differed from their native forms. On the other hand, the impact of phenolic compounds on mycelial growth is not correlated to the effect on laccase activity. Instead, mycelial growth and relative specific laccase activity appear to be modulated independently. All phenolic compounds showed not only inhibitory but also inductive effects on fungal growth and/or laccase activity, an observation which is reported for the first time. The simultaneous inhibition of growth and laccase activity demonstrated may serve as a basis for the development of a natural botryticide. Yet, the results showed considerable differences between genetically distinguishable strains, impeding the use of a specific phenolic compound against the genetic variety of wild strains. The present findings might have important implications for future understanding of Botrytis cinerea infections and sustainable Botrytis management including the role of polyphenols., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Kim Marie Umberath reports financial support was provided by German Ministry of Economics and Technology. Louis Backmann reports financial support was provided by German Ministry of Economics and Technology. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Botrytis cinerea is the phytopathogenic fungus responsible for the gray mold disease that affects crops worldwide. Essential oils (EOs) have emerged as a sustainable tool to reduce the adverse impact of synthetic fungicides. Nevertheless, the scarce information about the physiological mechanism action and the limitations to applying EOs has restricted its use. This study focused on elucidating the physiological action mechanisms and prospection of lipid nanoparticles to apply EO of Mentha piperita. The results showed that the EO of M. piperita at 500, 700, and 900 μL L -1 inhibited the mycelial growth at 100 %. The inhibition of spore germination of B. cinerea reached 31.43 % at 900 μL L -1 . The EO of M. piperita decreased the dry weight and increased pH, electrical conductivity, and cellular material absorbing OD 260 nm of cultures of B. cinerea. The fluorescence technique revealed that EO reduced hyphae width, mitochondrial activity, and viability, and increased ROS production. The formulation of EO of M. piperita loaded- solid lipid nanoparticles (SLN) at 500, 700, and 900 μL L -1 had particle size ∼ 200 nm, polydispersity index < 0.2, and stability. Also, the thermogravimetric analysis indicated that the EO of M. piperita-loaded SLN has great thermal stability at 50 °C. EO of M. piperita-loaded SLN reduced the mycelial growth of B. cinerea by 70 %, while SLN formulation (without EO) reached 42 % inhibition. These results supported that EO of M. piperita-loaded SLN is a sustainable tool for reducing the disease produced by B. cinerea., Competing Interests: Declaration of Competing Interest None., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

Botrytis cinerea is an important fungal pathogen that causes gray mold disease in plants. Previously, Bacillus velezensis TCS001 live culture presented broad-spectrum antifungal activity against various plant pathogenic fungi and oomycetes, particularly B. cinerea . Here, the bioactivity of lipopeptides produced by TCS001 against B. cinerea was investigated. The IC 50 values of the crude lipopeptide extract (CLE) from TCS001 to suppress mycelial growth and conidial germination were 14.20 and 49.39 mg/L, respectively. SEM and TEM imaging revealed that CLE caused morphological deformities and ultrastructural changes in the mycelium. Transcriptomic analyses combined with Δ Bcpsd mutant construction demonstrated that the CLE could confer antifungal activity via suppressing Bcpsd expression in the pathogen. In addition, the CLE activated the plant immune system by increasing the content of defense-related enzymes and the expression of marker genes in immunity signaling pathways in cucumber plants. Therefore, TCS001 CLE could be potentially developed into biopesticides for the biocontrol of gray mold disease.

Botrytis cinerea is a necrotrophic phytopathogen able to attack more than 200 different plant species causing strong yield losses worldwide. Many synthetic fungicides have been developed to control this disease, resulting in the rise of fungicide-resistance B. cinerea strains. The aim of this study was to identify Streptomyces strains showing antagonistic activity against B. cinerea to contribute to plant protection in an environmentally friendly way. We isolated 15 Actinomycete strains from 9 different Swiss soils. The culture filtrates of three isolates showing antifungal activity inhibited spore germination and delayed mycelial growth of B. cinerea . Infection experiments showed that Arabidopsis thaliana plants were more resistant to this pathogen after leaf treatment with the Streptomyces filtrates. Bioassay-guided isolation of the active compounds revealed the presence of germicidins A and B as well as of oligomycins A, B, and E. While germicidins were mostly inactive, oligomycin B reduced the mycelial growth of B. cinerea significantly. Moreover, all three oligomycins inhibited this fungus' spore germination, suggesting that these molecules might contribute to the Streptomyces 's ability to protect plants against infection by the broad host-pathogen Botrytis cinerea ., Importance: This study reports the isolation of new Streptomyces strains with strong plant-protective potential mediated by their production of specialized metabolites. Using the broad host range pathogenic fungus Botrytis cinerea , we demonstrate that the cell-free filtrate of selected Streptomyces isolates efficiently inhibits different developmental stages of the fungus, including mycelial growth and the epidemiologically relevant spore germination. Beyond in vitro experiments, the strains and their metabolites also efficiently protected plants against the disease caused by this pathogen. This work further identifies oligomycins as active compounds involved in the observed antifungal activity of the strains. This work shows that we can harness the natural ability of soil-borne microbes and of their metabolites to efficiently fight other microbes responsible for significant crop losses. This opens the way to the development of environmentally friendly health protection measures for crops of agronomical relevance, based on these newly isolated strains or their metabolic extracts containing oligomycins., Competing Interests: The authors declare no conflict of interest.

Botrytis cinerea is a plant pathogen causing the gray mold disease in a plethora of host plants. The control of the disease is based mostly on chemical pesticides, which are responsible for environmental pollution, while they also pose risks for human health. Furthermore, B. cinerea resistant isolates have been identified against many fungicide groups, making the control of this disease challenging. The application of biocontrol agents can be a possible solution, but requires deep understanding of the molecular mechanisms in order to be effective. In this study, we investigated the multitrophic interactions between the biocontrol agent Bacillus subtilis MBI 600, a new commercialized biopesticide, the pathogen B. cinerea and their plant host. Our analysis showed that this biocontrol agent reduced B. cinerea mycelial growth in vitro, and was able to suppress the disease incidence on cucumber plants. Moreover, treatment with B. subtilis led to induction of genes involved in plant immunity. RNA-seq analysis of B. cinerea transcriptome upon exposure to bacterial secretome, showed that genes coding for MFS and ABC transporters were highly induced. Deletion of the Bcmfs1 MFS transporter gene, using a CRISP/Cas9 editing method, affected its virulence and the tolerance of B. cinerea to bacterial secondary metabolites. These findings suggest that specific detoxification transporters are involved in these interactions, with crucial role in different aspects of B. cinerea physiology., (Copyright © 2021 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

Aureobasidium strains isolated from diverse unconventional environments belonging to the species A. pullulans, A. melanogenum, and A. subglaciale were evaluated for Volatile Organic Compounds (VOCs) production as a part of their modes of action against Botrytis cinerea of tomato and table grape. By in vitro assay, VOCs generated by the antagonists belonging to the species A. subglaciale showed the highest inhibition percentage of the pathogen mycelial growth (65.4%). In vivo tests were conducted with tomatoes and grapes artificially inoculated with B. cinerea conidial suspension, and exposed to VOCs emitted by the most efficient antagonists of each species (AP1, AM10, AS14) showing that VOCs of AP1 (A. pullulans) reduced the incidence by 67%, partially confirmed by the in vitro results. Conversely, on table grape, VOCs produced by all the strains did not control the fungal incidence but were only reducing the infection severity (< 44.4% by A. pullulans; < 30.5% by A. melanogenum, and A. subglaciale). Solid-phase microextraction (SPME) and subsequent gas chromatography coupled to mass spectrometry identified ethanol, 3-methyl-1-butanol, 2-methyl-1-propanol as the most produced VOCs. However, there were differences in the amounts of produced VOCs as well as in their repertoire. The EC 50 values of VOCs for reduction of mycelial growth of B. cinerea uncovered 3-methyl-1-butanol as the most effective compound. The study demonstrated that the production and the efficacy of VOCs by Aureobasidium could be directly related to the specific species and pathosystem and uncovers new possibilities for searching more efficient VOCs producing strains in unconventional habitats other than plants.

Pyraclostrobin (PYR) is a commonly used strobilurin fungicide, which inhibits mitochondrial respiration at the ubiquinol oxidation center site of the cytochrome bc1 complex. Little information is available regarding the crystal structure of PYR on its fungicidal effect. In this study, the crystal structures of eight PYRs (PYR-A to H) from different sources are determined by using high-resolution X-ray powder diffraction (XRPD) and model construction with the Pawley refinement module. The effects of PYRs on mycelium growth, the kinetics of mycelial growth, conidial germination, and tube elongation of conidia of Botrytis cinerea from tomato are compared. The level of organic acids in the mitochondrial tricarboxylic acid cycle of PYR-treated B. cinerea is analyzed. The results show that PYR-A to PYR-H have their own unique character of XRPD patterns, but the crystal morphology of eight PYRs presents in the triclinic crystal system and space group P 1̅. PYR-D with the eclipsed conformation and rational edge angles α (72.599°) and β (98.612°) in the crystal cell shows the highest inhibitory effect against mycelium growth with EC 50 as 3.383 μg mL -1 , the best time-dependent effects on the mycelium growth kinetics, and the strongest inhibition on tube elongation of conidia, whereas PYR-E with anticonformation is the worst. Moreover, a significant accumulation of fumarate, malate, and oxalate in the PYR-D-treated mycelium is observed. These findings reinforce the need for a definite crystal structure of PYR to limit usage and mitigate future selection pressure for gray mold management.

Grey mold is one of the most serious and catastrophic diseases, causing significant yield losses in fruits and vegetables worldwide. Iprodione is a broad spectrum agrochemical used as a foliar application as well as a seed protectant against many fungal and nematode diseases of fruits and vegetables from the last thirty years. The extensive use of agrochemicals produces resistance in plant pathogens and is the most devastating issue in food and agriculture. However, the molecular mechanism (whole transcriptomic analysis) of a resistant mutant of B. cinerea against iprodione is still unknown. In the present study, mycelial growth, sporulation, virulence, osmotic potential, cell membrane permeability, enzymatic activity, and whole transcriptomic analysis of UV (ultraviolet) mutagenic mutant and its wild type were performed to compare the fitness. The EC 50 (half maximal effective concentration that inhibits the growth of mycelium) value of iprodione for 112 isolates of B. cinerea ranged from 0.07 to 0.87 µg/mL with an average (0.47 µg/mL) collected from tomato field of Guangxi Province China. Results also revealed that, among iprodione sensitive strains, only B67 strain induced two mutants, M0 and M1 after UV application. The EC 50 of these induced mutants were 1025.74 μg/mL and 674.48 μg/mL, respectively, as compared to its wild type 1.12 μg/mL. Furthermore, mutant M0 showed higher mycelial growth sclerotia formation, virulence, and enzymatic activity than wild type W0 and M1 on potato dextrose agar (PDA) medium. The bctubA gene in the mutant M0 replaced TTC and GAT codon at position 593 and 599 by TTA and GAA, resulting in replacement of phenyl alanine into leucine (transversion C/A) and aspartic acid into glutamic acid (transversion T/C) respectively. In contrast, in bctubB gene, GAT codon at position 646 is replaced by AAT and aspartic acid converted into asparagine (transition G/A). RNA sequencing of the mutant and its wild type was performed without (M0, W0) and with iprodione treatment (M-ipro, W-ipro). The differential gene expression (DEG) identified 720 unigenes in mutant M-ipro than W-ipro after iprodione treatment (FDR ≤ 0.05 and log2FC ≥ 1). Seven DEGs were randomly selected for quantitative real time polymerase chain reaction to validate the RNA sequencing genes expression (log fold 2 value). The gene ontology (GO) enrichment and Kyoto encyclopedia genes and genomes (KEGG) pathway functional analyses indicated that DEG's mainly associated with lysophopholipase, carbohydrate metabolism, amino acid metabolism, catalytic activity, multifunctional genes (MFO), glutathione-S transferase (GST), drug sensitivity, and cytochrome P450 related genes are upregulated in mutant type (M0, M-ipro) as compared to its wild type (W0, W-ipro), may be related to induce resistant in mutants of B. cinerea against iprodione.

Fungal pathogens lead to severe quality deterioration and yield loss, making it urgent to explore efficient measures to control fungal diseases at the preharvest and postharvest stages of plants. Therefore, studies on natural substances targeting alternative antimicrobial targets have become hot spots of research. Here, we show that honokiol, a polyphenolic compound obtained from Magnolia officinalis, significantly suppressed mycelial growth and reduced virulence of B. cinerea on harvested fruit by inducing autophagic activities and apoptosis. Moreover, honokiol was capable of abolishing the mitochondrial membrane potential and inducing the accumulation of reactive oxygen species. Some key genes involved in pathogenicity on fruit were also found significantly down-regulated. In summary, honokiol was effective as an alternative agent targeting autophagic and apoptotic machineries to control the incidence of gray mold, which may further enrich the toolkit of crop managers for fighting postharvest diseases caused by this and similar fungi., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Aims: This study aimed to compare the effects of cold plasma (CP), ultraviolet (UV-C) and aqueous ozone (AO) on Botrytis cinerea and explore their application in preserving blueberry., Methods and Results: The effects of CP, UV-C or AO on B. cinerea were investigated. Results showed that three treatments effectively inhibited the growth of B. cinerea, increasing cell membrane penetrability and causing the leakage of cytoplasm and nucleic acid. Scanning electron microscopy showed that CP caused the mycelium fold and collapse depression; UV-C caused the mycelium shrivelled; mycelium treated with AO appeared to fold and break. In the in vivo test in blueberry, all treatments effectively inhibited the growth of micro-organisms, maintained qualities and enhanced antioxidant activities in blueberry during postharvest storage., Conclusion: Cold plasma, UV-C and AO effectively inhibited the growth of B. cinerea and significantly extended the shelf life of blueberry. Based on in vitro and in vivo tests, CP showed better effects to preserve blueberry compared with other treatments., Significance and Impact of the Study: Our findings revealed the effective mechanisms of CP, UV-C and AO to inhibit B. cinerea in vitro. CP exhibited better application potential to preserve fresh fruit than traditional sterilization methods such as UV-C and AO., (© 2019 The Society for Applied Microbiology.)

Crude extracts of Vitis vinifera canes represent a natural source of stilbene compounds with well characterized antifungals properties. In our trials, exogenous application of a stilbene extract (SE) obtained from grape canes on grapevine leaves reduces the necrotic lesions caused by Botrytis cinerea. The SE showed to possess a direct antifungal activity by inhibiting the mycelium growth. The activation of some grapevine defense mechanism was also investigated. H 2 O 2 production and activation of mitogen-activated protein kinase (MAPK) phosphorylation cascades as well as accumulation of stilbenoid phytoalexins were explored on grapevine cell suspension. Moreover, the transcription of genes encoding for proteins affecting defense responses was analyzed on grapevine plants. The SE induced some grapevine defense mechanisms including MAPK activation, and the expression of pathogenesis-related (PR) genes and of a gene encoding the glutathione-S-transferase 1 ( GST1) . By contrast, treatment of grapevine leaves with SE negatively regulates de novo stilbene production.

Background: The fungal pathogen Botrytis cinerea infects a broad range of horticultural plants worldwide, resulting in significant economic losses. A derivative of chitosan, oligochitosan, has been reported to be an eco-friendly alternative to synthetic fungicides., Results: Oligochitosan can greatly inhibit B. cinerea spore germination and induce protein carbonylation. To further investigate the molecular mechanism underlying the inhibitory effect, a comparative proteome analysis was conducted of oligochitosan-treated versus non-treated B. cinerea spores. The cellular proteins were obtained from B. cinerea spore samples and subjected to two-dimensional gel electrophoresis. In total, 21 differentially expressed proteins (DEPs) were identified. Three DEPs were up-regulated in the oligochitosan-treated versus the untreated spores, including scytalone dehydratase and a serine carboxypeptidase III precursor. By contrast, seven DEPs, including Hsp 88 and cell division cycle protein 48, were down-regulated by oligochitosan treatment. Notably, 10 DEPs, including phosphatidylserine decarboxylase proenzyme and ATP-dependent molecular chaperone HSC82, were only detected in the control spores, whereas one DEP, a non-annotated predicted protein, was only detected in the oligochitosan-treated spores., Conclusion: Oligochitosan may affect the spore germination of B. cinerea by impairing protein function. These findings have practical implications with respect to the use of oligochitosan for controlling fungal pathogens. © 2018 Society of Chemical Industry., (© 2018 Society of Chemical Industry.)

The effect of 8,8-dimethyl-3-[( R -phenyl)amino]-1,4,5(8 H )-naphthalentrione derivatives (compounds 1 ⁻ 13 ) on the mycelial growth of Botrytis cinerea was evaluated. The fungitoxic effect depended on the substituent and its position in the aromatic ring. Compounds substituted with halogens in meta and/or para positions (compounds 3 , 4 , 5 and 7 ), methyl (compounds 8 and 9 ), methoxyl (compounds 10 and 11 ), or ethoxy-carbonyl groups (compound 12 ) presented higher antifungal activity than compound 1 , which had an unsubstituted aromatic ring. In addition, compounds with halogens in the ortho position, such as compounds 2 and 6 , and a substitution with an acetyl group in the para position (compound 13 ) were less active. The role of the ABC efflux pump Bctr B-type as a defense mechanism of B. cinerea against these naphthalentrione derivatives was analyzed. This pump could be involved in the detoxification of compounds 2 , 6 , and 13 . On the contrary, this mechanism would not participate in the detoxification of compounds 1 , 7 , 9 and 12 . Finally, the biotransformation of compound 7 by B. cinerea was studied. A mixture of two biotransformed products was obtained. One of them was compound 7A , which is reduced at C1 and C4, compared to compound 7 . The other product of biotransformation, 7B, is oxidized at C7.

Bacillus sp. 6256 is a good biocontrol agent against Botrytis cinerea which caused tomato gray mold disease. Strain 6256 was identified as B. amyloliquefaciens by analysis of its partial gyrB gene sequence. To identify and characterize the antimycotic peptides from the culture broth of the bacterium, the antimicrobial substances produced by B. amyloliquefaciens 6256 were isolated by ammonium sulfate precipitation, Superdex 200 gel filtration chromatography and DEAE anion exchange chromatography. The purified compound was designated as P657. The biological activity of P657 was stable at as high as 100 °C for 20 min and in pH value ranged from 5 to 10. The antimycotic compound was resistant to trypsin and proteinase K, and could completely inhibit spore germination of Botrytis cinerea in vitro. MALDI-TOF-MS analysis results showed the presence of fengycins A (C 16 -C 17 ) and fengycins B (C 15 -C 17 ) isoforms in P657., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Botrytis cinerea is the pathogen of gray mold disease affecting a wide range of plant hosts, with consequential economic losses worldwide. The increased frequency of fungicide resistance of the pathogen challenges its disease management, and thus the development of alternative control strategies are urgently required. In this study, we showed excellent synergistic interactions between resveratrol and pyrimethanil. Significant synergistic values were recorded by the two-drug combination on the suppression of mycelial growth and conidia germination of B. cinerea . The combination of resveratrol and pyrimethanil caused malformation of mycelia. Moreover, the inoculation assay was conducted on table grape and consistent synergistic suppression of the two-drug combination was found in vivo. Our findings first revealed that the combination of resveratrol and pyrimethanil has synergistic effects against resistant B. cinerea and support the potential use of resveratrol as a promising adjuvant on the control of gray mold.

Botrytis cinerea has a high risk of developing resistance to fungicides. Fludioxonil, belonging to phenylpyrroles, has been used for more than three decades, however, only few cases of field resistance against phenylpyrroles have been reported. In this study, the highly fludioxonil-resistant (HR) isolates of Botrytis cinerea were firstly detected in the commercial greenhouses of strawberry in China in 2015, and biochemical characterization differences in high fludioxonil-resistance from strawberry and cucumber were compared. All of the five HR isolates from greenhouses of strawberry and cucumber could grow on PDA amended with 100 μg/mL fludioxonil, and exhibited a positive correlation between the resistance of dicarboximide fungicides and fludioxonil. Sporulation and sclerotium production of the strawberry-originated HR isolates were increased in comparison with the cucumber-originated HR isolates. No matter how the HR isolates were from strawberry and cucumber, all the HR isolates showed enhanced sensitivity to the osmotic agents, but with significant difference. Based on sequence alignment of the BcOS1 which codes protein bound by fludioxonil, two genotypes of the strawberry-originated HR isolates were observed, i.e., (F127S + I365N + S426P) and (G538R + A1259T), which were totally different from those of the cucumber-originated HR isolates. Molecular docking of fludioxonil to the binding site of BcOS1 protein from the five HR isolates illustrated that all the HR isolates had less affinity than the sensitive isolates. Our data indicated that genotypes of the HR isolates match the corresponding fludioxonil-selection pressure on the field populations of B. cinerea in the commercial greenhouses of the two host plants., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Extracts obtained from liquid mycelial fermentations of the Chilean fungus Stereum hirsutum (Sh134-11) showed antifungal activity against Botrytis cinerea. Two types of extracts were obtained: EtOAc-extract (liquid phase) and MeOH-extract (mycelial phase). Plate diffusion assay showed that EtOAc-extracts were more active than MeOH-extracts. A large-scale fermentation of Sh134-11 and chromatographic methods allowed to isolated four compounds: MS-3, Vibralactone, Vibralactone B and Sterenin D. Only Sterenin D showed antifungal activity against B. cinerea in the tests performed. Effects on the mycelial growth of B. cinerea showed that Sterenin D showed inhibition at 1000-2000 μg/mL reaching 67% and 76% respectively. Sterenin D was more effective to control the sporogenesis, inhibiting in 96% the sporulation at 500 μg/mL. Assays showed that Sterenin D exhibited a minimal fungicidal concentration (MFC) of 50 μg/mL and minimal inhibitory concentration (MIC) at 20 μg/mL. Our study indicated that submerged fermentations of Chilean S. hirsutum (Sh134-11) produced extracts with antifungal activity and Sterenin D is responsible for this activity, which could be used as possible biofungicides alternative to synthetic fungicides., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

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 EC 50 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 EC 50 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., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

β-carbolines (βCs) are alkaloids widely distributed in nature that have demonstrated antimicrobial properties. Here, we tested in vitro six βCs against Penicillium digitatum and Botrytis cinerea, causal agents of postharvest diseases on fruit and vegetables. Full aromatic βCs (harmine, harmol, norharmane and harmane) exhibited a marked inhibitory effect on conidia germination at concentrations between 0.5 and 1 mM, while dihydro-βCs (harmalina and harmalol) only caused germination delay. Harmol showed the highest inhibitory effect on both fungal pathogens. After 24 h of exposure to 1 mM harmol, conidia revealed a severe cellular damage, exhibiting disorganized cytoplasm and thickened cell wall. Harmol antimicrobial effect was fungicidal on B. cinerea, while it was fungistatic on P. digitatum. Conidia membrane permeabilization was detected in treatments with harmol at sub-inhibitory and inhibitory concentrations, for both pathogens. In addition, residual infectivity of P. digitatum on lemons and B. cinerea on blueberries was significantly reduced after exposure to this alkaloid. It also inhibited mycelial growth, preventing sporulation at the highest concentration tested. These results indicate that harmol might be a promising candidate as a new antifungal molecule to control causal agents of fruit diseases., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

In the attempt to reduce the negative impacts of chemical pesticides on environment and consumer's health, a new plant treatment practice minimizing the amount of pesticides needed during pests and diseases treatments has been developed. Our approach is based on combining the biocide effects of fungicide with the peroxyacetic acid (PAA) one. In this paper, we focused on the in vitro study of the antifungal activity of this combination against Botrytis cinerea, the most redoubtable threat of tomatoes plants in Morocco. First, different concentrations of a peroxyacetic acid product (PERACLEAN ® 5) and two commercially available fungicides SWITCH and SIGNUM were tested separately for their inhibitory effects on the mycelial growth and spores germination of B. cinerea. 100% inhibition of fungal growth was achieved using 16.77 and 14.47 μg/ml of SIGNUM and SWITCH respectively and 1.5% of PERACLEAN ® 5. When combined with 0.5% of the peroxyacetic acid mixture (PERACLEAN ® 5), the pesticides 100% effective concentrations decreased to 0.5 μg/ml for both pesticides. Hence, this approach allowed us to suppress the pathogen while minimizing the amounts of applied fungicides by more than 95%., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Botrytis cinerea is a model plant-pathogenic fungus that causes grey mould and rot diseases in a wide range of agriculturally important crops. A previous study has identified two enzymes and corresponding genes (bcdh, bcer) that are involved in the biochemical transformation of uridine diphosphate (UDP)-glucose, the major fungal wall nucleotide sugar precursor, to UDP-rhamnose. We report here that deletion of bcdh, the first biosynthetic gene in the metabolic pathway, or of bcer, the second gene in the pathway, abolishes the production of rhamnose-containing glycans in these mutant strains. Deletion of bcdh or double deletion of both bcdh and bcer has no apparent effect on fungal development or pathogenicity. Interestingly, deletion of the bcer gene alone adversely affects fungal development, giving rise to altered hyphal growth and morphology, as well as reduced sporulation, sclerotia production and virulence. Treatments with wall stressors suggest the alteration of cell wall integrity. Analysis of nucleotide sugars reveals the accumulation of the UDP-rhamnose pathway intermediate UDP-4-keto-6-deoxy-glucose (UDP-KDG) in hyphae of the Δbcer strain. UDP-KDG could not be detected in hyphae of the wild-type strain, indicating fast conversion to UDP-rhamnose by the BcEr enzyme. The correlation between high UDP-KDG and modified cell wall and developmental defects raises the possibility that high levels of UDP-KDG result in deleterious effects on cell wall composition, and hence on virulence. This is the first report demonstrating that the accumulation of a minor nucleotide sugar intermediate has such a profound and adverse effect on a fungus. The ability to identify molecules that inhibit Er (also known as NRS/ER) enzymes or mimic UDP-KDG may lead to the development of new antifungal drugs., (© 2016 BSPP AND JOHN WILEY & SONS LTD.)

2-Allylphenol (2-AP) is an effective fungicide against a number of plant pathogens, which can be metabolized and bio-transformed to four chemical compounds by Rhizoctonia cerealis. To determine if its degradation affects antifungal activity, two major metabolites derived from 2-AP including 2-(2-hydroxypropyl) phenol and 2-(3-hydroxypropyl) phenol were synthesized. Inhibition of mycelial growth of several plant pathogens by the metabolites was evaluated, and structures of two metabolites were determined by hydrogen nuclear magnetic resonance ( 1 H NMR). Among these metabolites, only 2-(2-hydroxypropyl) phenol inhibited test pathogens effectively. EC 50 values of 2-(2-hydroxypropyl) phenol for inhibition of mycelial growth of R. cerealis, Pythium aphanidermatum, Valsa mali and Botrytis cinerea ranged from 1.0 to 23.5μg/ml, which were lower than the parental fungicide 2-AP that ranged from 8.2 to 48.8μg/ml. Hyphae of R. cerealis and P. aphanidermatum treated with 2-(2-hydroxypropyl) phenol were twisted. Newly developed hyphae were slender, twisted and swollen on the tip, while old hyphae were hollow and ruptured. This is the first report indicating the formation of 2-(2-hydroxypropyl) phenol may have contributed to toxicity of 2-allylphenol in control of plant pathogens., (Copyright © 2016. Published by Elsevier Inc.)

Natural stilbenes (especially resveratrol) play important roles in plant protection by acting as both constitutive and inducible defenses. However, their exogenous applications on crops as fungicidal agents are challenged by their oxidative degradation and limited availability. In this study, a new class of resveratrol-inspired oxadiazole-stilbene hybrids was synthesized via Wittig-Horner reaction. Bioassay results indicated that some of the compounds exhibited potent fungicidal activity against Botrytis cinerea in vitro. Among these stilbene hybrids, compounds 11 showed promising inhibitory activity with the EC50 value of 144.6 μg/mL, which was superior to that of resveratrol (315.6 μg/mL). Remarkably, the considerably abnormal mycelial morphology was observed in the presence of compound 11. The inhibitory profile was further proposed by homology modeling and molecular docking studies, which showed the possible interaction of resveratrol and oxadiazole-stilbene hybrids with the cytochrome P450-dependent sterol 14α-demethylase from B. cinerea (BcCYP51) for the first time. Taken together, these results would provide new insights into the fungicidal mechanism of stilbenes, as well as an important clue for biology-oriented synthesis of stilbene hybrids with improved bioactivity against plant pathogenic fungi in crop protection.

Three phenotypes were detected in 161 Botrytis cinerea field isolates, including Zox(S)Car(S) (sensitive to zoxamide and carbendazim), Zox(S)Car(R) (sensitive to zoxamide and resistant to carbendazim), and Zox(R)Car(R) (resistant to zoxamide and carbendazim), but not Zox(R)Car(S) (resistant to zoxamide and sensitive to carbendazim). The baseline sensitivity to zoxamide was determined with a mean EC50 of 0.76 μg/ml. Two stable Zox(R)Car(S) isolates were obtained with a resistance factor of 13.28 and 20.43; there was a fitness penalty in mycelial growth rate, sporulation, virulence and sclerotium production. The results suggest that the resistance risk of B. cinerea to zoxamide is low where benzimidazoles have not been used. E198V, E198K and M233I, were detected in the β-tubulin of Zox(S)Car(R), Zox(R)Car(R), Zox(R)Car(S), respectively. Molecular docking indicated that position 198 in β-tubulin were targets for both zoxamide and carbendazim. The mutations at 198 prevented formation of hydrogen bonds between β-tubulin and carbendazim (E198V/K), and changed the conformation of the binding pocket of zoxamide (E198K). M233I had no effect on the binding of carbendazim but resulted in loss of a hydrogen bond between zoxamide and F200. M233 is suggested to be a unique target site for zoxamide and be very important in the function of β tubulin.

To develop new agents against strawberry grey mould and to aid in the development of biological pesticides, we investigated the inhibitory effect of a natural compound, phenazine-1-carboxamide (PCN), against Botrytis cinerea using a growth rate assay. Additionally, indoor toxicity and the in vitro control effect of PCN were further studied to determine its potential mechanisms of action on B. cinerea. PCN was inhibitory against B. cinerea with a 50% effective concentration (EC50) of 108.12 μg/mL; the toxicity of PCN was equivalent to that of carbendazim (CBM). The best in vitro control effect of PCN against grey mould in strawberry (fruit) reached 75.32%, which was slightly higher than that of CBM. The field control effect of PCN against grey mould reached a maximum of 72.31% at a PCN concentration of 700 μg/mL, which was 1.02 times higher than that of CBM. Fungistatic activity was observed at low concentrations of PCN, while high concentrations of PCN resulted in fungicidal activity against B. cinerea. This natural compound strongly inhibited both spore and sclerotium germination of B. cinerea, with the best relative inhibition rates of 77.03% and 82.11%, respectively. The inhibitory effect of PCN on mycelial growth of B. cinerea was significant and reached levels of 87.32%. Scanning electron microscopy observations revealed that after 48 h of PCN treatment, the mycelia appeared loose, locally twisted, and folded, with exudation of contents; the mycelia was withered and twisted, with edge burrs, deformations, ruptures and a sheet-like structure. Transmission electron microscopy observations revealed that after 48 h of PCN treatment, the structure of the cell nucleus was unclear and the vacuoles had ruptured; additionally, various organelles exhibited disordered structures, there were substantial non-membrane transparent inclusions, the cells were plasmolysed, the cell walls were collapsed in some cases, and the hyphal tissue was essentially necrotic. A PCN dosage of 35-140 μg/mL had no effect on the cell membrane permeability of the mycelia, while a PCN dosage of 700 μg/mL resulted in significant permeability. PCN inhibited B. cinerea toxin; the mycotoxin level was approximately 0.41 of the value recorded for the control at a PCN dosage of 700 μg/mL. PCN affected the activity of pectin methylgalacturonase (PMG), polygalacturonase (PG), cellulase (Cx) and β-glucosidase (BG); the lowest activities of PMG, PG, BG and Cx reached 0.3 U/mg, 0.62 U/mg, 0.64 U/mg, and 0.79 U/mg, respectively, after treatment with 700 μg/mL PCN.

The inhibitory effects on the mycelial growth of plant pathogen Botritys cinerea have been evaluated for a series of geranylphenols substituted with one, two and three methoxy groups in the aromatic ring. The results show that the antifungal activity depends on the structure of the geranylphenols, increasing from 40% to 90% by increasing the number of methoxy groups. On the other hand, the acetylation of the -OH group induces a change of activity that depends on the number of methoxy groups. The biological activity of digeranyl derivatives is lower than that exhibited by the respective monogeranyl compound. All tested geranylphenols have been synthesized by direct coupling of geraniol and the respective phenol. The effect of solvent on yields and product distribution is discussed. For monomethoxyphenols the reaction gives better yields when acetonitrile is used as a solvent and AgNO3 is used as a secondary catalyst. However, for di- and trimethoxyphenols the reaction proceeds only in dioxane.

The mechanism of action of the diterpenoid 3β-hydroxykaurenoic acid and the anthraquinone 4,4-dimethylanthracene-1,9,10(4H)-trione on the phytopathogenic fungus Botrytis cinerea was studied. The effect of both compounds on the respiratory process and on the membrane integrity of B. cinerea was evaluated. The results showed that 3β-hydroxykaurenoic acid inhibited the growth of this fungus by disrupting the plasmatic membrane. This compound also partially affected oxygen consumption of B. cinerea germinating conidia. Conversely, 4,4-dimethylanthracene-1,9,10(4H)-trione did not produce membrane disruption of B. cinerea. The effect of this compound on mycelial growth was notably increased by the presence of an inhibitor of the cyanide-resistant respiration pathway. It also was shown that the anthraquinone inhibited oxygen consumption by about 80%; therefore this compound would act as a potent inhibitor of the cytochrome pathway of the respiratory chain to exert its antifungal effect., (© 2015 by The Mycological Society of America.)

This study was undertaken to describe purification, identification, and characteristics of protocatechuic acid (PCA) isolated for the first time from Paenibacillus elgii HOA73 against Botrytis cinerea (the cause of gray mold disease on strawberry fruit). PCA was purified by different chromatographic techniques and identified as PCA (3,4-dihydroxybenzoic acid) by nuclear magnetic resonance and liquid chromatography-mass spectrometry analyses. PCA displayed potent antifungal activity against B. cinerea and Rhizoctonia solani. However, the antifungal activities were not sufficient to inhibit mycelial growth of Phytophthora capsici and Fusarium oxysporum. The minimum inhibitory concentration of PCA to inhibit any visible mycelial growth of both B. cinerea and R. solani was 64 µg ml(-1) . Most B. cinerea conidia displayed altered shape and absence of germination, or were degraded after treatment with 50 and 100 µg ml(-1) PCA, respectively. Moreover, gray mold formation on strawberry fruit was almost or completely inhibited by these PCA concentrations 7 days following infection with B. cinerea conidia, respectively. PCA may be a promising alternative to chemical fungicides as a potential biofungicide to prevent growth of B. cinerea in strawberry fruit disease management., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Inositol phosphorylceramide (IPC) synthase is the key enzyme with highly conserved sequences, which is involved in fungal sphingolipid biosynthesis. The antibiotic aureobasidin A (AbA) induces the death of fungi through inhibiting IPC synthase activity. The mutations of AUR1 gene coding IPC synthase in fungi and protozoa causes a resistance to AbA. However, the mechanism of AbA resistance is still elusive. In this paper, we generated two mutants of Botrytis cinerea with AbA-resistance, BcAUR1a and BcAUR1b, through UV irradiation. BcAUR1a lost an intron and BcAUR1b had three amino acid mutations (L197P, F288S and T323A) in the AUR1 gene. AbA strongly inhibits the activity of IPC synthase in wild-type B. cinerea, which leads to distinct changes in cell morphology, including the delay in conidial germination, excessive branching near the tip of the germ tube and mycelium, and the inhibition of the mycelium growth. Further, AbA prevents the infection of wild-type B. cinerea in tomato fruits via reducing oxalic acid secretion and the activity of cellulase and pectinase. On the contrary, AbA has no effect on the growth and pathogenicity of the two mutants. Although both mutants show a similar AbA resistance, the molecular mechanisms might be different between the two mutants.

In in vitro tests with 18 plant pathogens, the fungicide 3-[5-(4-chlorophenyl)-2,3-dimethyl-3-isoxazolidinyl] pyridine (SYP-Z048) was highly effective on inhibiting mycelial growth of various ascomycota and basidiomycota, with EC50 values ranging from 0.008 to 1.140 μg/ml. SYP-Z048 had much weaker activity against growth of oomycota with EC50 values > 100 μg/ml. In a second in vitro test with Monilinia fructicola isolates, SYP-Z048 inhibited mycelial growth (EC50 = 0.013 μg/ml), germ tube elongation (EC50 = 0.007 μg/ml), and sporulation but did not affect spore germination. In a detached pear fruit assay inoculated with M. fructicola isolates, SYP-Z048 showed protective and curative activity. Field tests indicated that SYP-Z048 was an efficacious fungicide for control of brown rot disease in two peach orchards. When applied to a single spot on a tomato leaflet in a compound leaf, SYP-Z048 suppressed the growth of Botrytis cinerea isolates on the rest 4 leaflets, indicating that the fungicide has systemic movement in plant tissues. These results indicate that SYP-Z048 has potential for management of brown rot causing by M. fructicola and other diseases caused by ascomycota and basidiomycota.

SYP-1620, a quinone-outside-inhibitor (QoI), is a novel broad-spectrum fungicide. In this study, 108 isolates of Botrytis cinerea from different geographical regions in Jiangsu Province of China were characterized for baseline sensitivity to SYP-1620. The curves of baseline sensitivity were unimodal with a mean EC50 value of 0.0130±0.0109 μg/mL for mycelial growth, 0.01147±0.0062 μg/mL for spore germination, respectively. The biological characterization of SYP-1620 against B. cinerea was determined in vitro. The results indicated that SYP-1620 has a strong inhibiting effect on spore germination, mycelial growth, and respiration. The protective and curative test of SYP-1620 suggested that protective effect was better than curative either on strawberry leaves or on cucumber leaves in vivo. In addition, the biological characterization of SYP-1620-resistant mutants of B. cinerea was investigated. SYP-1620 has no cross-resistance with other types of fungicide. Compared to the sensitive isolates, the resistant mutants had lower mycelial growth and virulence but not differ in mycelial dry weight. Sequencing indicated that SYP-1620 resistance was associated with a single point mutation (G143A) in the cytochrome b gene., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Bis(2,3-dibromo-4,5-dihydroxybenzyl) ether (BDDE) is a bromophenol isolated from marine algae. Previous reports have shown that BDDE possesses cytotoxic and antibacterial activity. In the present study, we demonstrate that BDDE displays broad-spectrum antifungal activities, especially on Botrytis cinerea. BDDE inhibits the growth of B. cinerea cultured on a solid medium of potato dextrose agar (PDA) as well as on the potato dextrose broth (PDB) medium. Moreover, BDDE decreases the incidence of fruit decay and severity of strawberries infected with B. cinerea. Further studies have revealed that BDDE decreases the germination rate and inhibits the mycelial growth of B. cinerea. The inhibition mechanisms are related to the disruption of the cell membrane integrity in B. cinerea spores and newly formed germ tubes. This study also suggests that BDDE possibly interacts with DNA via intercalation and minor groove binding. The studies provide evidence that BDDE has potential application in the control of gray mold after fruit harvest and the compound could serve as a candidate or lead template for rational drug design and for the development of antifungal agents.

Aimed at developing novel fungicides for relieving the ever-increasing pressure of agricultural production caused by phytopathogenic fungi, 28 new hydrazone derivatives of carabrone, a natural bioactive sesquisterpene, in three types were designed, synthesized and their antifungal activities against Botrytis cinerea and Colletotrichum lagenarium were evaluated. The result revealed that all the derivatives synthesized exhibited considerable antifungal activities in vitro and in vivo, which led to the improved activities for carabrone and its analogues and further confirmed their potential as antifungal agents.

Natural geranyl compounds are known to exhibit important biological activities. In this work a series of geranylphenols were synthesized to evaluate their effect on the mycelial growth of Botrytis cinerea. Geranyl derivatives were synthesized by direct geranylation reactions between the corresponding phenol derivatives and geraniol, using BF3.OEt2 as catalyst and AgNO3 as secondary catalyst. Previously reported molecules [geranylhydroquinone (2), geranylhydroquinone diacetate (6) and geranylphloroglucinol (9)], and new substances [(E)-4-(3,7-dimethylocta-2,6-dienyl)benzene-1,2,3-triol (geranyl-pyrogallol, 7), (E)-4-(3,7-dimethylocta-2,6-dienyl)benzene-1,2,3-triyl triacetate (8), (E)-2-(3,7-dimethylocta-2,6-dienyl)benzene-1,3,5-triyl triacetate geranylphloroglucinol triacetate (10), 2,4-bis((E)-3,7-dimethylocta-2,6-dienyl)benzene-1,3,5-triyl triacetate (11), 2,6-bis((E)-3,7-dimethylocta-2,6-dienyl)-3,5-dihydroxyphenyl acetate (12)], were obtained. All compounds were characterized by IR, HRMS and NMR spectroscopic data. The inhibitory effect of the synthesized compounds on the mycelial growth of Botrytis cinerea was tested in vitro. Excepting compound 11, all substances constrained the mycelial growth of Botrytis cinerea. The antifungal activity depends on the chemical structure of geranylphenol derivatives. Compounds 2 and 9 were the more effective substances showing inhibition degrees higher than those obtained with the commercial fungicide Captan, even at lower concentrations. Monosubstitution on the aromatic nucleus by a geranyl chain seems to be more effective for the inhibition of mycelial growth than a double substitution. These results suggest that the new derivatives of geranylphenols have the ability to block the mycelial development of the plant pathogen B. cinerea and that this capacity depends strongly on the structural features and lipophilicity of the compounds.

Eighteen N-substituted phenyl-2-acyloxycyclohexylsulfonamides (III) were designed and synthesized by the reaction of N-substituted phenyl-2-hydroxyl- cycloalkylsulfonamides (I, R(1)) with acyl chloride (II, R(2)) in dichloromethane under the catalysis of TMEDA and molecular sieve. High fungicidal active compound N-(2,4,5-trichlorophenyl)-2-(2-ethoxyacetoxy) cyclohexylsulfonamide (III-18) was screened out. Mycelia growth assay against the Botrytis cinerea exhibited that EC50 and EC80 of compound III-18 were 4.17 and 17.15 μg mL(-1) respectively, which was better than the commercial fungicide procymidone (EC50 = 4.46 μg mL(-1) and EC80 = 35.02 μg mL(-1)). For in vivo activity against B. cinerea in living leaf of cucumber, the control effect of compound III-18 was better than the fungicide cyprodinil. In addition, this new compound had broader fungicidal spectra than chlorothalonil.

Botrytis cinerea Pers, the gray mold fungus, is among the most dangerous plant pathogens that cause great losses in agricultural crops. The botryticidal activities of nanosized silver (nano Ag), fungal chitosan (CTS) irradiated fungal chitosan (IrCTS), and nano Ag-IrCTS composite were investigated. All of the examined materials exhibited powerful antifungal activity against B. cinerea. The most effective compound was nano Ag-IrCTS composite with a minimal inhibitory concentration of 125 μg/mL. The microscopic examination, of treated B. cinerea with the composite, revealed that an obvious alteration in mycelial shape was appeared accompanied and moderate lysis in fungal hyphae. With the prolongation of treatment, most of the fungal mycelia were lysed into small and elastic fragments. The consequence of strawberries coating, with antifungal composite based solution, was the disappearance of gray mold infection signs in 90% of the contaminated fruits after 7 d of storage, treated fruits had a fresh-like appearance at the end of storage. Coating with nano Ag-IrCTS solution could be highly recommended regarding its efficiency in prohibiting B. cinerea growth, preventing gray mold decay and enhancing the overall quality of coated strawberry fruits., (© 2013 Institute of Food Technologists®)

Aims: Tea tree oil (TTO) has been confirmed in previous study as a potential natural antifungal agent to control Botrytis cinerea and grey mould in fresh fruit. However, the mechanism of its action has not been clearly revealed, and some hypotheses mainly depended on the results obtained from the bacterial test. For the antifungal mechanism, the effect of TTO on the mycelium morphology and ultrastructure, cell wall and membrane, and membrane fatty acid composition of B. cinerea was investigated in vitro experiments., Methods and Results: Tea tree oil in vapour or contact phase exhibited higher activity against the mycelial growth of B. cinerea. Observations using scanning electron microscope and transmission electron microscope revealed that the mycelial morphology and ultrastructure alternations caused by TTO are the markedly shriveled or flatted empty hyphae, with thick cell walls, ruptured plasmalemma and cytoplasmic coagulation or leakage. Furthermore, TTO caused significantly higher alkaline phosphatase activity after 4-h treatment and markedly higher absorbance at 260 nm and electric conductivity in the external hyphae of fungi after 16-h treatment. Moreover, decreased unsaturated/saturated fatty acid ratio of the fungal membrane was also observed after TTO treatment., Conclusions: The methodology used in this study confirmed that the cell wall destroyed firstly in the presence of TTO, and then the membrane fatty acid composition changed, which resulted in the increasing of membrane permeability and releasing of cellular material. The above findings may be the main reason for TTO's antifungal ability to B. cinerea., Significance and Impact of the Study: Understanding the mechanism of TTO antifungal action to B. cinerea is helpful for its commercial application on the preservation of fresh fruit and vegetables., (© 2013 The Society for Applied Microbiology.)

This study demonstrates that the isoprenoid farnesol produces a toxic effect on the phytopathogenic fungus Botrytis cinerea in solid and liquid media. In solid media farnesol retarded 72 h the beginning of mycelial growth. Also, it was demonstrated that the toxic effect is due to farnesol triggers apoptosis in B. cinerea because ROS accumulation, DNA fragmentation and phosphatidylserine externalization were detected in farnesol-treated mycelium. Therefore, compounds that increase the intracellular farnesol or induce apoptosis could have a potential application as fungicide against B. cinerea.

Background: Succinate dehydrogenase inhibitors (SDHIs) constitute a fungicide class with increasing relevance in crop protection. These fungicides could play a crucial role in successful management of grey mould disease. In the present study the effect of fluopyram, a novel SDHI fungicide, on several developmental stages of Botrytis cinerea was determined in vitro, and the protective and curative activity against the pathogen was determined on strawberry fruit. Furthermore, fungal baseline sensitivity was determined in a set of 192 pathogen isolates., Results: Inhibition of germ tube elongation was found to be the most sensitive growth stage affected by fluopyram, while mycelial growth was found to be the least sensitive growth stage. Fluopyram provided excellent protective activity against B. cinerea when applied at 100 µg mL(-1) 96, 48 or 24 h before the artificial inoculation of the strawberry fruit. Similarly, fluopyram showed a high curative activity when it was applied at 100 µg mL(-1) 24 h post-inoculation, but, when applications were conducted 48 or 96 h post-inoculation, disease control efficacy was modest or low. The measurement of baseline sensitivity showed that it was unimodal in all the populations tested. The individual EC(50) values for fluopyram ranged from 0.03 to 0.29 µg mL(-1). In addition, no correlation was found between sensitivity to fluopyram and sensitivity to other fungicides, including cyprodinil, fenhexamid, fludioxonil, iprodione, boscalid and pyraclostrobin., Conclusions: The obtained biological activity, baseline sensitivity and cross-resistance relationship data suggest that fluopyram could play a key role in grey mould management in the near future and encourage its introduction into spray programmes., (Copyright © 2011 Society of Chemical Industry.)

The control of grapevine pathogens is a rising concern in Vitis vinifera culture. The current international trend is toward banning chemicals that are highly toxic to the environment and human workers, and adopting tighter regulations. We evaluated the impact of saponins on three kinds of organisms found in grapevine culture. The ectoparasitic nematode Xiphinema index, the parasitic fungus Botrytis cinerea and various yeast strains representative of the must fermentation population were incubated on synthetic media supplemented with variable concentrations of Quillaja saponaria saponins. Saponins induced reduction in the growth of B. cinerea and showed nematicide effects on X. index. The control of X. index and Botrytis cinerea is discussed in the context of the potential use of these chemicals as environmentally-friendly grapevine treatments. With Saccharomyces cerevisiae and other yeasts, saponins showed higher toxicity against S. cerevisiae strains isolated from wine or palm wine whereas laboratory strains or strains isolated from oak exhibited better resistance. This indicates that Q. saponaria saponins effects against yeast microflora should be assessed in the field before they can be considered an environmentally-safe new molecule against B. cinerea and X. index.

A study was conducted to identify volatile organic compounds or volatiles produced by Candida intermedia strain C410 using gas chromatography-mass spectrometry, and to determine efficacy of the volatiles of C. intermedia in suppression of conidial germination and mycelial growth of Botrytis cinerea and control of Botrytis fruit rot of strawberry. Results showed that, among 49 volatiles (esters, alcohols, alkenes, alkanes, alkynes, organic acids, ketones, and aldehydes) identified from C. intermedia cultures on yeast extract peptone dextrose agar, two compounds, 1,3,5,7-cyclooctatetraene and 3-methyl-1-butanol, were the most abundant. Synthetic chemicals of 1,3,5,7-cyclooctatetraene; 3-methyl-1-butanol; 2-nonanone; pentanoic acid, 4-methyl-, ethyl ester; 3-methyl-1-butanol, acetate; acetic acid, pentyl ester; and hexanoic acid, ethyl ester were highly inhibitory to conidial germination and mycelial growth of B. cinerea. Inhibition of conidial germination and mycelial growth of B. cinerea by volatiles of C. intermedia was also observed. Meanwhile, results showed that incidence and severity of Botrytis fruit rot of strawberry was significantly (P < 0.01) reduced by exposure of the strawberry fruit to the volatiles from C. intermedia cultures or C. intermedia-infested strawberry fruit. These results suggest that the volatiles of C. intermedia C410 are promising biofumigants for control of Botrytis fruit rot of strawberry.

The response regulator protein is a core element of two-component signaling pathway. In this study, we investigated functions of BRRG-1 of Botrytis cinerea, a gene that encodes a putative response regulator protein, which is homologous to Rrg-1 in Neurospora crassa. The BRRG-1 gene deletion mutant ΔBrrg1-62 was unable to produce conidia. The mutant showed increased sensitivity to osmotic stress mediated by NaCl and KCl, and to oxidative stress generated by H(2)O(2). Additionally, the mutant was more sensitive to the fungicides iprodione, fludioxonil, and triadimefon than the parental strain. Western-blot analysis showed that the Bos-2 protein, the putative downstream component of Brrg-1, was not phosphorylated in the ΔBrrg1-62. Real-time polymerase chain reaction assays showed that expression of BOS-2 also decreased significantly in the mutant. All of the defects were restored by genetic complementation of the ΔBrrg1-62 with the wild-type BRRG-1 gene. Plant inoculation tests showed that the mutant did not show changes in pathogenicity on rapeseed leaves. These results indicated that Brrg-1 is involved in the regulation of asexual development, sensitivity to iprodione, fludioxonil, and triadimefon fungicides, and adaptation to osmotic and oxidative stresses in B. cinerea.

The antifungal effect of three furyl compounds closely related to resveratrol, (E)-3,4,5-trimethoxy-β-(2-furyl)-styrene (1), (E)-4-methoxy-β-(2-furyl)-styrene (2) and (E)-3,5-dimethoxy-β-(2-furyl)-styrene (3) against Botrytis cinerea was analyzed. The inhibitory effect, at 100 µg ml(-1) of compounds 1, 2, 3 and resveratrol on conidia germination, was determined to be about 70%, while at the same concentration pterostilbene (a dimethoxyl derivative of resveratrol) produced complete inhibition. The title compounds were more fungitoxic towards in vitro mycelial growth than resveratrol and pterostilbene. Compound 3 was the most active and a potential explanation of this feature is given using density functional theory (DFT) calculations on the demethoxylation/demethylation process. Compound 3 was further evaluated for its effects on laccase production, oxygen consumption and membrane integrity of B. cinerea. An increase of the laccase activity was observed in the presence of compound 3 and, using Sytox Green nucleic acid stain, it was demonstrated that this compound altered B. cinerea membrane. Finally, compound 3 partially affected conidia respiration.

To explore new potential fungicides, a series of novel compounds, including 11 2-oxocycloalkylsulfonamide (3) and 21 2-hydroxycycloalkylsulfonamide (4) derivatives, were synthesized and their structures were confirmed by (1)H nuclear magnetic resonance (NMR), infrared (IR), and elemental analysis. The results of the bioassay showed that the compounds 3 and 4 possessed excellent fungicidal activity against Botrytis cinerea Pers. both in vitro and in vivo. The fungicidal activity of the compounds with 7- or 8-membered rings is better than those with 5-, 6-, or 12-membered rings. According to the results of the mycelium growth rate test, the EC50 values of the compounds 3C, 4C, 3D, and 4D were 0.80, 0.85, 1.22, and 1.09 μg/mL, respectively, and similar to or better than commercial fungicide procymidone. The bioassay results of spore germination indicated that most of the compounds exhibited obvious inhibitory effects against B. cinerea and the inhibition rates of 2-oxocycloalkylsulfonamides were higher than 2-hydroxycycloalkylsulfonamides, among them. The EC50 values of compounds 3A, 3B17, 3E, and 4A were 4.21, 4.21 3.24, and 5.29 μg/mL, respectively. Those compounds containing 5- or 6-membered rings showed better activity than those containing 7-, 8-, or 12-membered rings. Furthermore, the results of the pot culture test showed that almost all of the compounds had effective control activity in vivo and 2-hydroxycycloalkylsulfonamides were obviously superior to 2-oxocycloalkylsulfonamides. The compounds 3E, 4C and 4D presented higher control efficacy than procymidone and pyrimethanil against gray mold disease on cucumber plants.

Aims: To investigate the effect of tea polyphenol (TP) and Hanseniaspora uvarum alone or in combination against Botrytis cinerea in grapes and to evaluate the possible mechanisms involved., Methods and Results: TP alone was effective in controlling grey mould in grape at all concentrations. TP at 0.5 and 1.0% in combination with H. uvarum (1 x 10(6) CFU ml(-1)) showed a lower infection rate of grey mould. TP at 0.01% or above significantly inhibited the spore germination of B. cinerea. TP at 0.1% showed inhibition ability on mycelium growth of B. cinerea. The addition of TP did not affect the growth of H. uvarum in vitro and significantly increased the population of H. uvarum in vivo., Conclusions: TP exhibited an inhibitory effect against B. cinerea and improved the biocontrol efficacy of H. uvarum. The inhibitory effects of spore germination and mycelial growth of B. cinerea and the increased populations of H. uvarum in vivo may be some of the important mechanisms of TP., Significance and Impact of the Study: The results suggested that TP alone or in combination with biocontrol agents has great potential in the commercial management of postharvest diseases of fruits.

Background: 2-Allylphenol is a registered fungicide in China to control fungal diseases on tomato, strawberry and apple. It is synthetic and structurally resembles the active ingredient ginkgol isolated from Ginkgo biloba L. bark. 2-Allylphenol has been used in China for 10 years. However, its biochemical mode of action remains unclear. An in vitro study was conducted on the biochemical mechanism of 2-allyphenol inhibiting Botrytis cinerea (Pers. ex Fr.)., Results: The inhibition was approximately 3 times stronger when the fungus was grown on non-fermentable source, glycerol, than that on a fermentable carbon source, glucose. Inhibition of B. cinerea and Magnaporthe oryzae (Hebert) Barr mycelial growth was markedly potentiated in the presence of salicylhydroxamic acid (SHAM), an inhibitor of mitochondrial alternative oxidase. Furthermore, at 3 h after treatment with 2-allylphenol, oxygen consumption had recovered, but respiration was resistant to potassium cyanide and sensitive to SHAM, indicating that 2-allylphenol had the ability to induce cyanide-resistant respiration. The mycelium inhibited in the presence of 2-allylphenol grew vigorously after being transferred to a fungicide-free medium, indicating that 2-allylphenol is a fungistatic compound. Adenine nucleotide assay showed that 2-allylphenol depleted ATP content and decreased the energy charge values, which confirmed that 2-allylphenol is involved in the impairment of the ATP energy generation system., Conclusion: These results suggested that 2-allylphenol induces cyanide-resistant respiration and causes ATP decrease, and inhibits respiration by an unidentified mechanism., ((c) 2009 Society of Chemical Industry.)

Aims: To characterize the volatile antifungal compound produced by Oxyporus latemarginatus EF069 and to examine in vitro and in vivo fumigation activity of the fungus., Methods and Results: An antifungal volatile-producing strain, O. latemarginatus EF069 inhibited the mycelial growth of Alternaria alternata, Botrytis cinerea, Colletotrichum gloeosporioides, Fusarium oxysporum f. sp. lycopersici, and Rhizoctonia solani by mycofumigation. An antifungal volatile compound was isolated from the hexane extract of wheat bran-rice hull cultures of O. latemarginatus EF069 by repeated silica gel column chromatography and identified as 5-pentyl-2-furaldehyde (PTF). The purified PTF inhibited mycelial growth of R. solani in a dose-dependent manner. The mycofumigation with solid cultures of EF069 also reduced effectively the development of postharvest apple decay caused by B. cinerea and Rhizoctonia root rot of moth orchid caused by R. solani., Conclusions: Oxyporus latemarginatus EF069 showed in vitro and in vivo fumigation activity against plant pathogenic fungi by producing 5-pentyl-2-furaldehyde., Significance and Impact of the Study: Oxyporus latemarginatus EF069 producing an antifungal volatile compound may be used as a biofumigant for the control of fungal plant diseases.

Ethylene production by infected plants is an early resistance response leading to activation of plant defense pathways. However, plant pathogens also are capable of producing ethylene, and ethylene might have an effect not only on the plant but on the pathogen as well. Therefore, ethylene may play a dual role in fungus-plant interactions by affecting the plant as well as the pathogen. To address this question, we studied the effects of ethylene on the gray mold fungus Botrytis cinerea and the disease it causes on Nicotiana benthamiana plants. Exposure of B. cinerea to ethylene inhibited mycelium growth in vitro and caused transcriptional changes in a large number of fungal genes. A screen of fungal signaling mutants revealed a Galpha null mutant (deltabcg1) which was ethylene insensitive, overproduced ethylene in vitro, and showed considerable transcriptional changes in response to ethylene compared with the wild type. Aminoethoxyvinylglycine (AVG)-treated, ethylene-nonproducing N. benthamiana plants developed much larger necroses than ethylene-producing plants, whereas addition of ethylene to AVG-treated leaves restricted disease spreading. Ethylene also affected fungal gene expression in planta. Expression of a putative pathogenicity fungal gene, bcspl1, was enhanced 24 h after inoculation in ethylene-producing plants but only 48 h after inoculation in ethylene-nonproducing plants. Our results show that the responses of B. cinerea to ethylene are partly mediated by a G protein signaling pathway, and that ethylene-induced plant resistance might involve effects of plant ethylene on both the plant and the fungus.