Efficacy of antifungal agents against fungal spores: An in vitro study using microplate laser nephelometry and an artificially infected 3D skin model.

Dermal fungal infections seem to have increased over recent years. There is further a shift from anthropophilic dermatophytes to a growing prevalence of zoophilic species and the emergence of resistant strains. New antifungals are needed to combat these fungi and their resting spores. This study aimed to investigate the sporicidal effects of sertaconazole nitrate using microplate laser nephelometry against the microconidia of Trichophyton, chlamydospores of Epidermophyton, blastospores of Candida, and conidia of the mold Scopulariopsis brevicaulis. The results obtained were compared with those from ciclopirox olamine and terbinafine. The sporicidal activity was further determined using infected three-dimensional full skin models to determine the antifungal effects in the presence of human cells. Sertaconazole nitrate inhibited the growth of dermatophytes, molds, and yeasts. Ciclopirox olamine also had good antifungal activity, although higher concentrations were needed compared to sertaconazole nitrate. Terbinafine was highly effective against most dermatophytes, but higher concentrations were required to kill the resistant strain Trichophyton indotineae. Sertaconazole nitrate, ciclopirox olamine, and terbinafine had no negative effects on full skin models. Sertaconazole nitrate reduced the growth of fungal and yeast spores over 72 h. Ciclopirox olamine and terbinafine also inhibited the growth of dermatophytes and molds but had significantly lower effects on the yeast. Sertaconazole nitrate might have advantages over the commonly used antifungals ciclopirox olamine and terbinafine in combating resting spores, which persist in the tissues, and thus in the therapy of recurring dermatomycoses.

The elemental defense effect of cadmium on Alternaria brassicicola in Brassica juncea.

BACKGROUND: The elemental defense hypothesis states a new defensive strategy that hyperaccumulators defense against herbivores or pathogens attacks by accumulating heavy metals. Brassica juncea has an excellent ability of cadmium (Cd) accumulation. However, the elemental defense effect and its regulation mechanism in B. juncea remain unclear. RESULTS: In this study, we profiled the elemental defense effect and the molecular regulatory mechanism in Cd-accumulated B. juncea after Alternaria brassicicola infection. B. juncea treated with 180 mg Kg- 1 DW CdCl2 2.5H2O exhibited obvious elemental defense effect after 72 h of infection with A. brassicicola. The expression of some defense-related genes including BjNPR1, BjPR12, BjPR2, and stress-related miRNAs (miR156, miR397, miR398a, miR398b/c, miR408, miR395a, miR395b, miR396a, and miR396b) were remarkably elevated during elemental defense in B. juncea. CONCLUSIONS: The results indicate that Cd-accumulated B. juncea may defend against pathogens by coordinating salicylic acid (SA) and jasmonic acid (JA) mediated systemic acquired resistance (SAR) and elemental defense in a synergistic joint effect. Furthermore, the expression of miRNAs related to heavy metal stress response and disease resistance may regulate the balance between pathogen defense and heavy metal stress-responsive in B. juncea. The findings provide experimental evidence for the elemental defense hypothesis in plants from the perspectives of phytohormones, defense-related genes, and miRNAs.

Electrohydraulic Streamer Discharge Plasma-Enhanced Alternaria brassicicola Disinfection in Seed Sterilization.

As emerging chemical-free and eco-friendly technologies, nonthermal (gas discharge) plasma and (liquid phase) plasma-activated water (PAW) offer exceptional microbial disinfection solutions for biological, medical, environmental, and agricultural applications. Herein, we present electrohydraulic streamer discharge plasma (ESDP), which combines streamer discharge plasma (SDP) and PAW generated at a gas-liquid interface, to sterilize Chinese kale (Brassica oleracea var. alboglabra) seeds contaminated with Alternaria brassicicola (A. brassicicola). The results showed that the ESDP treatment of A. brassicicola-inoculated seeds provides a ∼75% reduction of A. brassicicola (incident percentage) compared with nontreated seeds. Likewise, the healthy seedling percentage of the plasma-treated seeds was significantly improved to ∼70%, while that of the nontreated seeds remained at ∼15%. A microscopic examination was performed, and it confirmed that ESDP can damage the A. brassicicola spores attached to Chinese kale seeds and lead to severe morphological abnormalities after treatment. Also, an electric field simulation was performed, and it indicated that the strongly localized electric field at the liquid-gas interface on the seed surface boundary had initiated local breakdown of the gas at the air-liquid interface, resulting in exceptional physical-chemical reactions for antimicrobial efficacy beyond typical plasma treatments. Moreover, the optical emission spectra and physicochemical properties (pH, conductivity, and oxidation-reduction potential) showed that inactivation is mainly associated with the reactive oxygen-nitrogen species in the liquid and gas phases. We believe that this work is of great interest when using electrical discharge plasma on liquid interfaces in food, agricultural, and medical industries.

Antifungal compound, methyl hippurate from Bacillus velezensis CE 100 and its inhibitory effect on growth of Botrytis cinerea.

Botrytis cinerea, the causal agent of gray mold is one of the major devastating fungal pathogens that occurs in strawberry cultivation and leads to massive losses. Due to the rapid emergence of resistant strains in recent years, an ecofriendly disease management strategy needs to be developed to control this aggressive pathogen. Bacillus velezensis CE 100 exhibited strong antagonistic activity with 53.05% against B. cinerea by dual culture method. In the present study, 50% of culture filtrate supplemented into PDA medium absolutely inhibited mycelial growth of B. cinerea whereas the highest concentration (960 mg/L) of different crude extracts including ethyl acetate, chloroform, and n-butanol crude extracts of B. velezensis CE 100, strongly inhibited mycelial growth of B. cinerea with the highest inhibition of 79.26%, 70.21% and 69.59% respectively, resulting in severe damage to hyphal structures with bulging and swellings. Hence, the antifungal compound responsible was progressively separated from ethyl acetate crude extract using medium pressure liquid chromatography. The purified compound was identified as methyl hippurate by nuclear magnetic resonance and mass spectrometry. The inhibitory effect of methyl hippurate on both spore germination and mycelial growth of B. cinerea was revealed by its dose-dependent pattern. The spore germination rate was completely restricted at a concentration of 3 mg/mL of methyl hippurate whereas no mycelial growth was observed in agar medium supplemented with 4 mg/mL and 6 mg/mL of methyl hippurate by poisoned food method. Microscopic imaging revealed that the morphologies of spores were severely altered by long-time exposure to methyl hippurate at concentrations of 1 mg/mL, 2 mg/mL and 3 mg/mL and hyphae of B. cinerea were severely deformed by exposure to methyl hippurate at concentrations of 2 mg/mL, 4 mg/mL and 6 mg/mL. No significant inhibition on tomato seed germination was observed in treatments with methyl hippurate (2 mg/mL) for both 6 h and 12 h soaking period as compared to the controls. Based on these results, B. velezensis CE 100 could be considered a potential agent for development of environmentally friendly disease control strategies as a consequence of the synergetic interactions of diverse crude metabolites and methyl hippurate.

A Unique Dual-Readout High-Throughput Screening Assay To Identify Antifungal Compounds with Aspergillus fumigatus.

Treatment of invasive mold infections is limited by the lack of adequate drug options that are effective against these fatal infections. High-throughput screening of molds using traditional antifungal assays of growth is problematic and has greatly limited our ability to identify new mold-active agents. Here, we present a high-throughput screening platform for use with Aspergillus fumigatus, the most common causative agent of invasive mold infections, for the discovery of novel mold-active antifungals. This assay detects cell lysis through the release of the cytosolic enzyme adenylate kinase and, thus, is not dependent on changes in biomass or metabolism to detect antifungal activity. The ability to specifically detect cell lysis is a unique aspect of this assay that allows identification of molecules that disrupt fungal cell integrity, such as cell wall-active molecules. We also found that germinating A. fumigatus conidia release low levels of adenylate kinase and that a reduction in this background allowed us to identify molecules that inhibit conidial germination, expanding the potential for discovery of novel antifungal compounds. Here, we describe the validation of this assay and proof-of-concept pilot screens that identified a novel antifungal compound, PIK-75, that disrupts cell wall integrity. This screening assay provides a novel platform for high-throughput screens with A. fumigatus for the identification of anti-mold drugs. IMPORTANCE Fungal infections caused by molds have the highest mortality rates of human fungal infections. These devastating infections are hard to treat and available antifungal drugs are often not effective. Therefore, the identification of new antifungal drugs with mold activity is critical. Drug screening with molds is challenging and there are limited assays available to identify new antifungal compounds directly with these organisms. Here, we present an assay suitable for use for high-throughput screening with a common mold pathogen. This assay has exciting future potential for the identification of new drugs to treat these fatal infections.

Impact of the physiological state of fungal spores on their inactivation by active chlorine and hydrogen peroxide.

This study aimed at assessing the impact of the physiological state of fungal spores on inactivation by sodium hypochlorite, 0.1% and 0.2% active chlorine, and 3% hydrogen peroxide. In this context, two physiological states were compared for 4 fungal species (5 strains). The first physiological state corresponded to fungal spores produced at 0.99 aw and harvested using an aqueous solution (laboratory conditions), while the second one corresponded to fungal spores produced under a moderate water stress (0.95 aw) and dry-harvested (mechanical harvesting without use of any water, mimicking food plant conditions). Aspergillus flavus "food plant" conidia were more resistant to all tested fungicide molecules than the "laboratory" ones. The same phenomenon was observed for Penicillium commune UBOCC-A-116003 conidia treated with hydrogen peroxide. However, this isolate did not exhibit any inactivation difference between "laboratory" and "food plant" conidia treated with sodium hypochlorite. Similarly, the physiological state of Cladosporium cladosporioides conidia did not impact the efficacy of the tested biocides. P. commune UBOCC-A-112059 "food plant" and "laboratory" conidia were more resistant to hydrogen peroxide and sodium hypochlorite, respectively. As for Mucor circinelloides, "laboratory" spores were more resistant to all disinfectant than the "food plant" ones. Noteworthy, regardless of the physiological state, all M. circinelloides and C. cladosporioides conidia were inactivated for 5 min treatment at 0.2% active chlorine and for 2.5 min treatment at 0.1% active chlorine, while the conidia of all the other species remained viable for these treatments. The obtained data indicate that the efficacy of disinfectant molecules depends not only on the encountered fungal species and its intraspecific diversity but also on the spore physiological state.

A Comparative Study of Antimicrobial and Antioxidant Activities of Plant Essential Oils and Extracts as Candidate Ingredients for Edible Coatings to Control Decay in 'Wonderful' Pomegranate.

This study determined the antimicrobial and antioxidant activity of lemongrass (LO), thyme (TO), and oregano (OO) essential oils and ethanolic extracts of pomegranate peel (PPE) and grape pomace (GPE) as candidate ingredients for edible coatings. Antifungal effects against Botrytis cinerea and Penicillium spp. were tested using paper disc and well diffusion methods. Radical scavenging activity (RSA) was evaluated using 2,2-diphenyl-1-picrylhydrazyl and 2,2'-azinobis-3-ethylbenzothiazoline-6-sulfonic acid assays. Gas chromatography-mass spectrometry analysis identified limonene (16.59%), α-citral (27.45%), ß-citral (27.43%), thymol (33.31%), paracymene (43.26%), 1,8-cineole (17.53%), and trans-caryphellene (60.84%) as major compounds of the essential oils. From both paper disc and well diffusion methods, LO recorded the widest zone of inhibition against tested microbes (B. cinerea and Penicillium spp.). The minimum inhibitory concentrations of LO against B. cinerea and Penicillium spp., were 15 µL/mL and 30 µL/mL, respectively. The highest (69.95%) and lowest (1.64%) RSA at 1 mg/mL were recorded for PPE and OO. Application of sodium alginate and chitosan-based coatings formulated with LO (15 or 30 µL/mL) completely inhibited spore germination and reduced the decay severity of 'Wonderful' pomegranate. Lemongrass oil proved to be a potential antifungal agent for edible coatings developed to extend shelf life of 'Wonderful' pomegranate.

Synergistic effect of ozone and chlorine on inactivating fungal spores: Influencing factors and mechanisms.

Effective control of fungal contamination in water is vital to provide healthy and safe drinking water for human beings. Although ozone was highly effective in inactivating fungi in water, it was limited by a lack of continuous disinfection ability in water supply system. In present study, the inactivation of fungal spores by combining ozone and chlorine was investigated. The synergistic effects of Aspergillus niger and Trichoderma harzianum spores reached 0.47- and 0.55-log within 10 min, respectively. The inactivation efficiency and the synergistic effect would be affected by disinfectant concentration, pH, and temperature. The combined inactivation caused more violent oxidative stimulation and more severe damage to the fungal spores than the individual inactivation based on the flow cytometry analysis and the scanning electron microscopy observation. The synergistic effect during the combined inactivation process was attributed to the generation of hydroxyl radicals by the reaction between ozone and chlorine and the promotion of chlorine penetration by the destruction of cell wall by ozone. The combined inactivation efficiency in natural water samples was reduced by 26.4-43.8% compared with that in PBS. The results of this study provided an efficient and feasible disinfection method for the control of fungi in drinking water.

Antifungal Activities of cis-trans Citral Isomers against Trichophyton rubrum with ERG6 as a Potential Target.

Trichophyton rubrum causes ringworm worldwide. Citral (CIT), extracted from Pectis plants, is a monoterpene and naturally composed of geometric isomers neral (cis-citral) and geranial (trans-citral). CIT has promising antifungal activities and ergosterol biosynthesis inhibition effects against several pathogenic fungi. However, no study has focused on neral and geranial against T. rubrum, which hinders the clinical application of CIT. This study aimed to compare antifungal activities of neral and geranial and preliminarily elucidate their ergosterol biosynthesis inhibition mechanism against T. rubrum. Herein, the disc diffusion assays, cellular leakage measurement, flow cytometry, SEM/TEM observation, sterol quantification, and sterol pattern change analyses were employed. The results showed geranial exhibited larger inhibition zones (p < 0.01 or 0.05), higher cellular leakage rates (p < 0.01), increased conidia with damaged membranes (p < 0.01) within 24 h, more distinct shriveled mycelium in SEM, prominent cellular material leakage, membrane damage, and morphological changes in TEM. Furthermore, geranial possessed more promising ergosterol biosynthesis inhibition effects than neral, and both induced the synthesis of 7-Dehydrodesmosterol and Cholesta-5,7,22,24-tetraen-3ß-ol, which represented marker sterols when ERG6 was affected. These results suggest geranial is more potent than neral against T. rubrum, and both inhibit ergosterol biosynthesis by affecting ERG6.

Optimization of granular formulations of Metarhizium humberi microsclerotia with humectants.

Granular microsclerotial formulations of entomopathogenic fungi deserve attention because of their post-application, in situ production of new conidia that enhance and prolong mycoinsecticidal efficacy against a target pest insect. Because high ambient moisture is a crucial condition to induce fungal development and conidiogenesis on granules, we tested the impacts of the additions of three humectants-glycerin, propylene glycol, and polyethylene glycol 400-on water absorption by pellets incorporating microsclerotia of Metarhizium humberi IP 46 with microcrystalline cellulose or vermiculite carriers, and on the production of infective conidia of IP 46 microsclerotia in ambient humidities suboptimal for routine conidiogenesis. Glycerin facilitated greater and faster absorption of water than the other humectants. Microcrystalline cellulose absorbed low quantities of water without any added humectant whereas vermiculite did not. IP 46 did not grow or sporulate on pellets prepared with or without glycerin at 86% relative humidity (RH) or on control pellets without glycerin at 91% RH; conidial production on pellets prepared with vermiculite or microcrystalline cellulose and 10% glycerin reached 1.1 × 105 conidia/mg and 1 × 105 conidia/mg, respectively, after 20 days of exposure at 91% RH. Hence, these results strongly support glycerin as a suitable humectant for granular microsclerotial formulations of this fungus.

Discovery of Fungus-Specific Targets and Inhibitors Using Chemical Phenotyping of Pathogenic Spore Germination.

There is a critical need for new antifungal drugs; however, the lack of available fungus-specific targets is a major hurdle in the development of antifungal therapeutics. Spore germination is a differentiation process absent in humans that could harbor uncharacterized fungus-specific targets. To capitalize on this possibility, we developed novel phenotypic assays to identify and characterize inhibitors of spore germination of the human fungal pathogen Cryptococcus. Using these assays, we carried out a high-throughput screen of ∼75,000 drug-like small molecules and identified and characterized 191 novel inhibitors of spore germination, many of which also inhibited yeast replication and demonstrated low cytotoxicity against mammalian cells. Using an automated, microscopy-based, quantitative germination assay (QGA), we discovered that germinating spore populations can exhibit unique phenotypes in response to chemical inhibitors. Through the characterization of these spore population dynamics in the presence of the newly identified inhibitors, we classified 6 distinct phenotypes based on differences in germination synchronicity, germination rates, and overall population behavior. Similar chemical phenotypes were induced by inhibitors that targeted the same cellular function or had shared substructures. Leveraging these features, we used QGAs to identify outliers among compounds that fell into similar structural groups and thus refined relevant structural moieties, facilitating target identification. This approach led to the identification of complex II of the electron transport chain as the putative target of a promising structural cluster of germination inhibitory compounds. These inhibitors showed high potency against Cryptococcus spore germination while maintaining low cytotoxicity against mammalian cells, making them prime candidates for development into novel antifungal therapeutics. IMPORTANCE Fungal pathogens cause 1.5 million deaths annually, and there is a critical need for new antifungal drugs. However, humans and fungi are very similar on a molecular level, and so many drugs that kill fungi also damage human cells, leading to extreme side effects, including death. The lack of fungus-specific targets is a major hurdle in the development of antifungal therapeutics. Spore germination is a process absent in humans that could harbor fungus-specific targets. To capitalize on this possibility, we developed new assays to identify and characterize inhibitors of spore germination of the human fungal pathogen Cryptococcus. Using these assays, we identified and characterized 191 novel inhibitors of spore germination. These inhibitors showed high potency against Cryptococcus spore germination while maintaining low cytotoxicity against mammalian cells, making them prime candidates for development into novel antifungal therapeutics.

Combination of supercritical CO2 and high-power ultrasound for the inactivation of fungal and bacterial spores in lipid emulsions.

For the first time, this study addresses the intensification of supercritical carbon dioxide (SC-CO2) treatments using high-power ultrasound (HPU) for the inactivation of fungal (Aspergillus niger) and bacterial (Clostridium butyricum) spores in oil-in-water emulsions. The inactivation kinetics were analyzed at different pressures (100, 350 and 550 bar) and temperatures (50, 60, 70, 80, 85 °C), depending on the microorganism, and compared to the conventional thermal treatment. The inactivation kinetics were satisfactorily described using the Weibull model. Experimental results showed that SC-CO2 enhanced the inactivation level of both spores when compared to thermal treatments. Bacterial spores (C.butyricum) were found to be more resistant to SC-CO2 + HPU, than fungal (A.niger) ones, as also observed in the thermal and SC-CO2 treatments. The application of HPU intensified the SC-CO2 inactivation of C.butyricum spores, e.g. shortening the total inactivation time from 10 to 3 min at 85 °C. However, HPU did not affect the SC-CO2 inactivation of A.niger spores. The study into the effect of a combined SC-CO2 + HPU treatment has to be necessarily extended to other fungal and bacterial spores, and future studies should elucidate the impact of HPU application on the emulsion's stability.

Transcription in fungal conidia before dormancy produces phenotypically variable conidia that maximize survival in different environments.

Fungi produce millions of clonal asexual conidia (spores) that remain dormant until favourable conditions occur. Conidia contain abundant stable messenger RNAs but the mechanisms underlying the production of these transcripts and their composition and functions are unknown. Here, we report that the conidia of three filamentous fungal species (Aspergillus nidulans, Aspergillus fumigatus, Talaromyces marneffei) are transcriptionally active and can synthesize mRNAs. We find that transcription in fully developed conidia is modulated in response to changes in the environment until conidia leave the developmental structure. Environment-specific transcriptional responses can alter conidial content (mRNAs, proteins and secondary metabolites) and change gene expression when dormancy is broken. Conidial transcription affects the fitness and capabilities of fungal cells after germination, including stress and antifungal drug (azole) resistance, mycotoxin and secondary metabolite production and virulence. The transcriptional variation that we characterize in fungal conidia explains how genetically identical conidia mature into phenotypically variable conidia. We find that fungal conidia prepare for the future by synthesizing and storing transcripts according to environmental conditions present before dormancy.

Antifungal Activity of Quinofumelin against Fusarium graminearum and Its Inhibitory Effect on DON Biosynthesis.

Fusarium graminearum, causal agent of Fusarium head blight (FHB), causes a huge economic loss. No information is available on the activity of quinofumelin, a novel quinoline fungicide, against F. graminearum or other phytopathogens. In this study, we used mycelial growth and spore germination inhibition methods to determine the inhibitory effect of quinofumelin against F. graminearum in vitro. The results indicated that quinofumelin excellently inhibited mycelial growth and spore germination of F. graminearum, with the average EC50 values of 0.019 ± 0.007 µg/mL and 0.087 ± 0.024 µg/mL, respectively. In addition, we found that quinofumelin could significantly decrease deoxynivalenol (DON) production and inhibit the expression of DON-related gene TRI5 in F. graminearum. Furthermore, we found that quinofumelin could disrupt the formation of Fusarium toxisome, a structure for producing DON. Western blot analysis demonstrated that the translation level of TRI1, a marker gene for Fusarium toxisome, was suppressed by quinofumelin. The protective and curative assays indicated that quinofumelin had an excellent control efficiency against F. graminearum on wheat coleoptiles. Taken together, quinofumelin exhibits not only an excellent antifungal activity on mycelial growth and spore germination, but also could inhibit DON biosynthesis in F. graminearum. The findings provide a novel candidate for controlling FHB caused by F. graminearum.

Functional Genomic and Biochemical Analysis Reveals Pleiotropic Effect of Congo Red on Aspergillus fumigatus.

Inhibition of fungal growth by Congo red (CR) has been putatively associated with specific binding to ß-1,3-glucans, which blocks cell wall polysaccharide synthesis. In this study, we searched for transcription factors (TFs) that regulate the response to CR and interrogated their regulon. During the investigation of the susceptibility to CR of the TF mutant library, several CR-resistant and -hypersensitive mutants were discovered and further studied. Abnormal distorted swollen conidia called Quasimodo cells were seen in the presence of CR. Quasimodo cells in the resistant mutants were larger than the ones in the sensitive and parental strains; consequently, the conidia of the resistant mutants absorbed more CR than the germinating conidia of the sensitive or parental strains. Accordingly, this higher absorption rate by Quasimodo cells resulted in the removal of CR from the culture medium, allowing a subset of conidia to germinate and grow. In contrast, all resting conidia of the sensitive mutants and the parental strain were killed. This result indicated that the heterogeneity of the conidial population is essential to promote the survival of Aspergillus fumigatus in the presence of CR. Moreover, amorphous surface cell wall polysaccharides such as galactosaminogalactan control the influx of CR inside the cells and, accordingly, resistance to the drug. Finally, long-term incubation with CR led to the discovery of a new CR-induced growth effect, called drug-induced growth stimulation (DIGS), since the growth of one of them could be stimulated after recovery from CR stress.IMPORTANCE The compound Congo red (CR) has been historically used for coloring treatment and histological examination as well to inhibit the growth of yeast and filamentous fungi. It has been thought that CR binds to ß-1,3-glucans in the fungal cell wall, disrupting the organization of the cell wall structure. However, other processes have been implicated in affecting CR sensitivity. Here, we explore CR susceptibility through screening a library of genetic null mutants. We find several previously uncharacterized genetic regulators important for CR susceptibility. Through biochemical and molecular characterization, we find cell membrane permeability to be important. Additionally, we characterize a novel cell type, Quasimodo cells, that occurs upon CR exposure. These cells take up CR, allowing the growth of the remaining fungi. Finally, we find that priming with CR can enhance long-term growth in one mutant.

Evaluation of the Effect of Strigolactones and Synthetic Analogs on Fungi.

Strigolactones (SLs) are components of root exudates as a consequence of active release from the roots into the soil. Notably, they have been described as stimulants of seed germination in parasitic plants and of the presymbiotic growth in arbuscular mycorrhizal (AM) fungi, which are a crucial component of the plant root beneficial microbiota. SLs have therefore the potential to influence other microbes that proliferate in the soil around the roots and may interact with plants. A direct effect of SL analogs on the in vitro growth of a number of saprotrophic or plant pathogenic fungi was indeed reported.Here we describe a standardized method to evaluate the effect of SLs or their synthetic analogs on AM and filamentous fungi. For AM fungi, we propose a spore germination assay since it is more straightforward than the hyphal branching assay and it does not require deep expertise and skills. For filamentous fungi that can grow in axenic cultures, we describe the assay based on SLs embedded in the solid medium or dissolved in liquid cultures where the fungus is inoculated to evaluate the effect on growth, hyphal branching or conidia germination. These assays are of help to test the activity of natural SLs as well as of newly designed SL analogs for basic and applied research.

A Simple Antifungal assay for Testing Actinomycetes and Other Microbial Extracts.

Antifungal assay in vitro is a useful tool for the characterization of biological activity of microbial extracts. Here we describe a simple in vitro test at two final extract concentrations that allows long-term storage of the plates containing dry extracts before using. The assay protocol is described for two fungal strains, a unicellular yeast, with clinical interest (Candida albicans), and a sporulated and phytopathogenic filamentous fungus (Botrytis cinerea). They could serve as models for adapting other filamentous/yeast-like fungi.Plates are prepared by placing 100 and 10 µg, respectively, of the organic extracts in microtiter 96-well plates, where the test will be performed. The assay develops by adding 200 µL of a spore suspension 104 spores/mL for B. cinerea and 106 cells/mL for C. albicans in Sabouraud medium.After the incubation of the plates at 25 °C, for 2 days for C. albicans and 5 days for B. cinerea , the growth of the fungal targets is evaluated in a plate reader for unicellular yeast , or visually under the microscope for filamentous fungi. If visually evaluated, observed growth can be assigned to different categories by comparison with growth control and inhibition control. Inhibition effect on C. albicans at eight concentrations of amphotericin B (8-4-2-1-0.5-0.25-0.125-0.00625 µg/mL) or B. cinerea exposed of eight concentrations of iprodione (100-50-10-5-1-0.5-0.1-0.05 µg/mL) are used as inhibition controls, respectively.

Chlorogenic acid induces ROS-dependent apoptosis in Fusarium fujikuroi and decreases the postharvest rot of cherry tomato.

Chlorogenic acid is a plant polyphenol with antioxidant and antimicrobial activities. Fusarium fujikuroi is a fungal pathogen that causes many vegetables and fruits, including tomato, to rot. The effects of chlorogenic acid on the development of Fusarium rot of cherry tomato fruit were examined in the present study. Results showed that conidial germination, germ tube elongation, cell viability, and mycelial growth of F. fujikuroi were all significantly inhibited by chlorogenic acid. Chlorogenic acid stimulated the accumulation of reactive oxygen species (ROS), leading to cell apoptosis in F. fujikuroi. The addition of N-acetylcysteine partially recovered the mycelial growth, implying the antifungal activity of chlorogenic acid is related to a ROS burst. The application of chlorogenic acid decreased disease incidence and severity in cherry tomato fruit in a concentration-dependent manner. Taken together, these results suggest that chlorogenic acid inhibits the postharvest rot of cherry tomato fruit caused by F. fujikuroi by inducing cellular oxidative stress in the pathogen.

The Inactivation by Curcumin-Mediated Photosensitization of Botrytis cinerea Spores Isolated from Strawberry Fruits.

Photosensitization is a novel environmentally friendly technology with promising applications in the food industry to extend food shelf life. In this study, the natural food dye curcumin, when combined with visible light (430 nm), was shown to be an effective photosensitizer against the common phytopathogenic fungi Botrytis cinerea (the cause of grey mould). Production of the associated phytotoxic metabolites botrydial and dihydrobotrydial was measured by our newly developed and validated HRAM UPLC-MS/MS method, and was also shown to be reduced by this treatment. With a light dose of 120 J/cm2, the reduction in spore viability was directly proportional to curcumin concentrations, and the overall concentration of both botrydial and dihydrobotrydial also decreased with increasing curcumin concentration above 200 µM. With curcumin concentrations above 600 µM, the percentage reduction in fungal spores was close to 100%. When the dye concentration was increased to 800 µM, the spores were completely inactive and neither botrydial nor dihydrobotrydial could be detected. These results suggest that curcumin-mediated photosensitization is a potentially effective method to control B. cinerea spoilage, and also to reduce the formation of these phytotoxic botryane secondary metabolites.

Influence of Mycosphaerella and Teratosphaeria leaf diseases on chemical composition of essential oils of Eucalyptus globulus and effect of these essential oils on ascospores germination.

In a first step, essential oils were extracted from Eucalyptus globulus leaves, healthy and with symptoms and signs of Mycosphaerella leaf disease (MLD) and Teratosphaeria leaf disease (TLD), in two leaf stages. Stage 1: sessile, oval leaves covered by a waxy layer of a bluish colour, with opposite phyllotaxis, inserted along stems of quadrangular section. Stage 2: narrow and sickle leaves with a greyish green surface, mainly on the abaxial surface, inserted in alternating pairs along rounded stems. The essential oils were extracted by hydrodistillation and were analyzed by gas chromatography coupled with mass spectrometry. Chemical composition data and percentages of essential oil constituents were submitted to cluster analysis and principal component analysis. In a second step, under in vitro conditions, was evaluated the germination of Teratosphaeria nubilosa (one of the causal agents of TLD) ascospores in contact with the four types of essential oils extracted. The evaluations were performed at 24, 48 and 72 h after the experiments were assembled. The present study made it possible to distinguish and identify the chemical composition of essential oils from the eucalypt leaves used, and allowed 1,8-cineole to be identified as the major component for the essential oils investigated. The contact between essential oils and T. nubilosa spores allowed to prove the inhibition of the ascospores germination, being more efficient for the essential oils extracted from materials with the disease, which presented high amounts of 1,8-cineole.