Endophytic Fungi Isolated from Baccharis linearis and Echinopsis chiloensis with Antifungal Activity against Botrytis cinerea.

Botrytis cinerea is one of the most important phytopathogens in agriculture worldwide, infecting economically important crops. The main control of this fungus is by synthetic fungicides, causing the selection of resistant isolates. Compounds produced by endophytic fungi have been shown to have antifungal activity against this pathogen and can be used as an alternative to synthetic fungicides. The aim of this work was to isolate endophytic fungi from Chilean foothills in the Metropolitan Region. Ten fungi were isolated from Echinopsis chiloensis and Baccharis linearis, however, only two isolates inhibited the mycelial growth of B. cinerea by antibiosis and were identified as Epicoccum sp. and Pleosporales sp. Extracts at 200 mg L-1 from Epicoccum sp. and Pleosporales sp. showed antifungal activity against B. cinerea of 54.6 and 44.6% respectively. Active compounds in the Epicoccum sp. extracts were mainly alkaloids and phenolic compounds; meanwhile, in the Pleosporales sp. extracts, terpenes and/or saponins were responsible for the antifungal activity.

Characterization of the Fungitoxic Activity on Botrytis cinerea of N-phenyl-driman-9-carboxamides.

A total of 12 compounds were synthesized from the natural sesquiterpene (-) drimenol (compounds 4 to 15). The synthesized compounds corresponded to N-phenyl-driman-9-carboxamide derivatives, similar to some fungicides that inhibit the electron-transport chain. Their structures were characterized and confirmed by 1H NMR, 13C NMR spectroscopy, and mass spectrometry. Compounds 5 to 15 corresponded to novel compounds. The effect of the compounds on the mycelial growth of Botrytis cinerea was evaluated. Methoxylated and chlorinated compounds in the aromatic ring (compounds 6, 7, 12, and 13) exhibited the highest antifungal activity with IC50 values between 0.20 and 0.26 mM. On the other hand, the effect on conidial germination of B. cinerea of one methoxylated compound (6) and one chlorinated compound (7) was analyzed, and no inhibition was observed. Additionally, compound 7 decreased 36% the rate of oxygen consumption by germinating conidia.

Endophytic Fungi Isolated from Plants Growing in Central Andean Precordillera of Chile with Antifungal Activity against Botrytis cinerea.

Botrytis cinerea is an important phytopathogenic fungus affecting the fruit production around the world. This fungus is controlled mainly by using synthetic fungicides, but many resistant isolates have been selected by the indiscriminate use of fungicides. Endophytic fungi or secondary metabolites obtained from them become an alternative method of control for this fungus. The aim of this work was to identify endophytic fungi with antifungal activity against the plant pathogenic fungus B. cinerea isolated from plants from Central Andean Precordillera of Chile. Three endophytic fungi (Ac1, Lc1 and Ec1) with antifungal activity against B. cinerea were isolated from native and endemic plants growing in Central Andean Precordillera of Chile. The isolates Lc1 (isolated from Lithraea caustica) and Ac1 (isolated from Acacia caven) were identified as Alternaria spp. and the isolate Ec1 (isolated from Echinopsis chiloensis) was identified as Aureobasidium spp. The isolated endophytic fungi would inhibit B. cinerea through the secretion of diffusible and volatile compounds affecting the mycelial growth, conidia germination and interestingly, it was also shown that the volatile compounds produced by the three isolated endophytic fungi suppressed the sporulation of B. cinerea.

Essential Oil, Extracts, and Sesquiterpenes Obtained From the Heartwood of Pilgerodendron uviferum Act as Potential Inhibitors of the Staphylococcus aureus NorA Multidrug Efflux Pump.

Staphylococcus aureus is a serious human pathogen that is highly adaptive to environmental conditions and rapidly develops antibiotic resistance. The use of efflux pumps to reduce antibiotic concentrations at the intracellular level is one of the main mechanisms by which bacteria develop antibiotic resistance. The management of efflux pumps, specifically NorA, which is expressed by S. aureus strains, is a valuable strategy for restoring susceptibility in strains resistant to antibacterial agents. In recent years, many studies have focused on searching for natural substances that can reverse efflux pump-mediated resistance in S. aureus. Extracts and compounds obtained from plants can be efficient efflux pump inhibitors (EPIs) and represent a potentially patient-friendly strategy for controlling S. aureus. In the present study, we evaluated the ability of essential oils, petroleum ether extracts, dichloromethane extract (DCME) and six compounds isolated from the heartwood of Pilgerodendron uviferum (Cupressaceae) and two synthetic derivatives to inhibit efflux in NorA pumps in the following three S. aureus strains: K2378, which overexpressed the norA gene (norA++), K1902 (norA-deleted, ΔnorA) and the parental strain, NCTC 8325-4. Efflux activity was evaluated using a fluorometric method that measured the accumulation of the universal efflux pump substrate ethidium bromide (EtBr). Only DCME and the compounds 15-copaenol and epi-cubenol inhibited EtBr efflux by K2378. Even the lowest concentration of 15-copaenol exhibited a stronger inhibitory effect than carbonyl cyanide m-chlorophenyl hydrazone on EtBr efflux by K2378. 15-copaenal only showed inhibition of EtBr efflux in K2378 cells at 125 µg/mL, but not superior to the control inhibitor and 15-copaenyl acetate exerted no intrinsic EPI activity against K2378. Fractional inhibitory concentration index (FICI) values obtained in the checkerboard assays, indicated that all combinations between DCME, epi-cubenol and 15-copaenol, and tested antibiotics showed a synergistic effect in wild type, norA ++ and ΔnorA strains. Moreover, those were not toxic for the HeLa cell line at concentrations in which the synergistic effect and inhibitory activity of efflux pumps was determined. Other extracts and compounds obtained from P. uviferum did not display EtBr efflux-inhibiting activity against the evaluated S. aureus strains.

Antifungal Activity against Botrytis cinerea of 2,6-Dimethoxy-4-(phenylimino)cyclohexa-2,5-dienone Derivatives.

In this work the enzyme laccase from Trametes versicolor was used to synthetize 2,6-dimethoxy-4-(phenylimino)cyclohexa-2,5-dienone derivatives. Ten products with different substitutions in the aromatic ring were synthetized and characterized using ¹H- and 13C-NMR and mass spectrometry. The 3,5-dichlorinated compound showed highest antifungal activity against the phytopathogen Botrytis cinerea, while the p-methoxylated compound had the lowest activity; however, the antifungal activity of the products was higher than the activity of the substrates of the reactions. Finally, the results suggested that these compounds produced damage in the fungal cell wall.

Detoxification Mechanism of 8,8-Dimethyl-3-[(R-phenyl)amino]-1,4,5(8H)-naphthalentrione Derivatives by Botrytis cinerea.

The effect of 8,8-dimethyl-3-[(R-phenyl)amino]-1,4,5(8H)-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.

Role of sfk1 Gene in the Filamentous Fungus Penicillium roqueforti.

The sfk1 (suppressor of four kinase) gene has been mainly studied in Saccharomyces cerevisiae, where it was shown to be involved in growth and thermal stress resistance. This gene is widely conserved within the phylum Ascomycota. Despite this, to date sfk1 has not been studied in any filamentous fungus. Previously, we found that the orthologous of sfk1 was differentially expressed in a strain of Penicillium roqueforti with an altered phenotype. In this work, we have performed a functional characterization of this gene by using RNAi-silencing technology. The silencing of sfk1 in P. roqueforti resulted in decreased apical growth and the promotion of conidial germination, but interesting, it had no effect on conidiation. In addition, the attenuation of the sfk1 expression sensitized the fungus to osmotic stress, but not to thermal stress. RNA-mediated gene-silencing of sfk1 also affected cell wall integrity in the fungus. Finally, the silencing of sfk1 depleted the production of the main secondary metabolites of P. roqueforti, namely roquefortine C, andrastin A, and mycophenolic acid. To the best of our knowledge this is the first study of the sfk1 gene in filamentous fungi.

Structure-Activity and Lipophilicity Relationships of Selected Antibacterial Natural Flavones and Flavanones of Chilean Flora.

In this study, we tested eight naturally-occurring flavonoids-three flavanones and five flavones-for their possible antibacterial properties against four Gram-positive and four Gram-negative bacteria. Flavonoids are known for their antimicrobial properties, and due their structural diversity; these plant-derived compounds are a good model to study potential novel antibacterial mechanisms. The lipophilicity and the interaction of antibacterial compounds with the cell membrane define the success or failure to access its target. Therefore, through the determination of partition coefficients in a non-polar/aqueous phase, lipophilicity estimation and the quantification of the antibacterial activity of different flavonoids, flavanones, and flavones, a relationship between these parameters was assessed. Active flavonoids presented diffusion coefficients between 9.4 × 10-10 and 12.3 × 10-10 m²/s and lipophilicity range between 2.0 to 3.3. Active flavonoids against Gram-negative bacteria showed a narrower range of lipophilicity values, compared to active flavonoids against Gram-positive bacteria, which showed a wide range of lipophilicity and cell lysis. Galangin was the most active flavonoid, whose structural features are the presence of two hydroxyl groups located strategically on ring A and the absence of polar groups on ring B. Methylation of one hydroxyl group decreases the activity in 3-O-methylgalangin, and methylation of both hydroxyl groups caused inactivation, as shown for 3,7-O-dimethylgalangin. In conclusion, the amphipathic features of flavonoids play a crucial role in the antibacterial activity. In these compounds, hydrophilic and hydrophobic moieties must be present and could be predicted by lipophilicity analysis.

Synergistic interactions between phenolic compounds identified in grape pomace extract with antibiotics of different classes against Staphylococcus aureus and Escherichia coli.

Synergy could be an effective strategy to potentiate and recover antibiotics nowadays useless in clinical treatments against multi-resistant bacteria. In this study, synergic interactions between antibiotics and grape pomace extract that contains high concentration of phenolic compounds were evaluated by the checkerboard method in clinical isolates of Staphylococcus aureus and Escherichia coli. To define which component of the extract is responsible for the synergic effect, phenolic compounds were identified by RP-HPLC and their relative abundance was determined. Combinations of extract with pure compounds identified there in were also evaluated. Results showed that the grape pomace extract combined with representatives of different classes of antibiotics as ß-lactam, quinolone, fluoroquinolone, tetracycline and amphenicol act in synergy in all S. aureus and E. coli strains tested with FICI values varying from 0.031 to 0.155. The minimal inhibitory concentration (MIC) was reduced 4 to 75 times. The most abundant phenolic compounds identified in the extract were quercetin, gallic acid, protocatechuic acid and luteolin with relative abundance of 26.3, 24.4, 16.7 and 11.4%, respectively. All combinations of the extract with the components also showed synergy with FICI values varying from 0.031 to 0.5 and MIC reductions of 4 to 125 times with both bacteria strains. The relative abundance of phenolic compounds has no correlation with the obtained synergic effect, suggesting that the mechanism by which the synergic effect occurs is by a multi-objective action. It was also shown that combinations of grape pomace extract with antibiotics are not toxic for the HeLa cell line at concentrations in which the synergistic effect was observed (47 µg/mL of extract and 0.6-375 µg/mL antibiotics). Therefore, these combinations are good candidates for testing in animal models in order to enhance the effect of antibiotics of different classes and thus restore the currently unused clinical antibiotics due to the phenomenon of resistance. Moreover, the use of grape pomace is a good and low-cost alternative for this purpose being a waste residue of the wine industry.

In vitro and in vivo evaluation of the antioxidant and prooxidant activity of phenolic compounds obtained from grape (Vitis vinifera) pomace.

The antioxidant and/or prooxidant ability of extracts obtained from wine waste were analyzed using in vitro and in vivo assays. Cyclic voltammetry was used as the in vitro assay to determine the antioxidant and/or prooxidant properties and, the in vivo effect on mycelial growth of the fungus Botrytis cinerea was evaluated. In addition, the prooxidant activity was evaluated by intracellular oxidation of compound 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) in B. cinerea. The extracts used in this study were obtained from grape pomace of Cabernet Sauvignon, Carménère and Syrah varieties from the Misiones de Rengo Vineyard by simple extraction, using methanol/HCl 1% (v/v), ethanol 70% (v/v), or Soxhlet extraction. According to the results obtained, gallic acid was the most represented phenolic compound independent of grape variety and extraction method. In addition, vanillic acid; protocatechuic acid, syringic acid, quercetin and kaempferol were found in the extracts. From this study it was possible concluded that, depending of the method of extraction of the grape residues and the grape variety (Cabernet Sauvignon, Carménère and Syrah), the extracts showed antioxidant and/or prooxidant activity. However, no correlation can be established between the anodic oxidation potentials of the extracts and their effect on the fungus B. cinerea.

Farnesol induces apoptosis-like phenotype in the phytopathogenic fungus Botrytis cinerea.

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.

Anthocyanin composition in aged Chilean Cabernet Sauvignon red wines.

Anthocyanins in aged Cabernet Sauvignon red wines were studied by HPLC-MS. The major anthocyanin in 6, 7, and 8year old red wine extracts was the dimer vitisin A produced by condensation of malvidin-3-O-glucoside mediated by pyruvic acid. In aged wine, the content of malvidin-3-O-glucoside decreased with age with a concomitant increase of vitisin A. The latter is accompanied by several condensation products with molecular weight up to 1500m/z. Differential pulse voltammetry indicated that aged wines have lower antioxidant capacity than young wine (400mV), but higher than malvidin-3-O-glucoside (483mV). Tafel's plots showed that the electrochemical process occurring in aged wines is different from young wines. Six, seven and eight year old wines show similar behaviour with plots of 234, 177 and 188mV/dec, respectively. These values are higher than the expected 120mV/dec corresponding to a first electronic transfer but smaller compared to the 523mV/dec corresponding to young wine.

In vitro antifungal activity of the diterpenoid 7 alpha-hydroxy-8(17)-labden-15-oic acid and its derivatives against Botrytis cinerea.

We investigated the inhibitory effect of the natural diterpenoids, 7alpha-hydroxy-8(17)-labden-15-oic acid (salvic acid, 1), 7alpha-acetanoyloxy-8(17)-labden-15-oic acid (acetylsalvic acid, 2) and the hemisynthetic diterpenoids 7alpha-acyloxy-8(17)-labden-15-oic acids derivatives, 7alpha-propanoyloxy-8(17)-labden-15-oic acid (propanoylsalvic acid, 3), 7alpha-butanoyloxy-8(17)-labden-15-oic acid (butanoylsalvic acid, 4) and 7alpha-isopentanoyloxy-8(17)-labden-15-oic acid (isopentanoylsalvic acid, 5), against Botrytis cinerea. Diterpenoid fungitoxicity was assessed using the radial growth test method. All diterpenoids, with the exception of isopentenoylsalvic acid, inhibited the mycelial growth of B. cinerea in solid media. Shortest side-chain diterpenoids were more effective than the derivatives with longer chains in the inhibition of B. cinerea mycelial growth. The results suggest that hydrophobicity and structural features would be important factors in the antifungal effect of these diterpenoids. Studies on a possible action mechanism of natural diterpenoids, salvic acid and acetylsalvic acid, showed that these diterpenoids exerted their effect by a different mechanism. Salvic acid did not alter cytoplasmic membrane or cause respiratory chain inhibition. Instead, acetylsalvic acid affected the cytoplasmic membrane producing leakage of 260-nm absorbing compounds.

The diterpenoid ent-16-kauren-19-oic acid acts as an uncoupler of the bacterial respiratory chain.

ent-16-Kauren-19-oic acid is a bacteriolytic diterpenoid present in the resin of the medicinal plant Pseudognaphalium vira vira. The compound and its methyl ester showed strong activity against gram-positive bacteria, whereas the derivative 3beta-hydroxy-ent-kauren-19-oic acid was inactive against all assayed bacteria at the maximal concentration used (250 microg/mL). The bacteriolytic effect of ent-16-kauren-19-oic acid (5 microg/mL) was confirmed with cultures of Bacillus cereus and Staphylococcus aureus, whereas the methyl derivative (12 microg/mL) showed only a bacteriostatic effect. Both compounds stimulated oxygen consumption and proton conduction of whole cells, as reflected by an abrupt increase in the extracellular pH. These results indicate that ent-16-kauren-19-oic acid acts as a respiratory chain uncoupler, and that this function is strongly affected by minor structural substitutions, suggesting a tight activity-structure relationship. The ultimate effect of the uncoupling mechanism demonstrated by ent-16-kauren-19-oic acid is bacterial lysis. The disruption of the bacterial membrane integrity caused by the diterpenoid compound was determined using SYTOX Green stain and visualized by fluorescence microscopy.

Effect of 13-epi-sclareol on the bacterial respiratory chain.

13-epi-sclareol is a labdane-type diterpene isolated from the resinous exudates of the medicinal plant species Pseudognaphalium cheiranthifolium (Lam.) Hilliard et Burtt. and P. heterotrichium (Phil.) A. Anderb. This compound has antibacterial activity only against Gram-positive bacteria, showing a bactericidal and lytic action. The interaction of 13- epi-sclareol with the bacterial respiratory chain was analyzed. The compound inhibited oxygen consumption of intact Gram-positive cells, but not with Gram-negative bacteria. The compound inhibited NADH oxidase and cytochrome c reductase activities, while coenzyme Q reductase and the cytochrome c oxidase activities were not affected. These results suggest that the target site of 13-epi-sclareol is located between coenzyme Q and cytochrome c. Using cytoplasmic membrane fractions, the results of the analysis of the enzyme activities associated with the respiratory chain complexes were the same for both Gram-positive and Gram-negative bacteria, indicating that the compound has no access to the cytoplasmic membrane of intact Gram-negative bacteria. Thus, the Gram-negative envelope may act as a physical barrier that prevents the access of this compound to the site of action.

Characterization of the antifungal activity on Botrytis cinerea of the natural diterpenoids kaurenoic acid and 3beta-hydroxy-kaurenoic acid.

The antifungal activity on Botrytis cinerea of the diterpenoids 3beta-hydroxy-kaurenoic acid and kaurenoic acid, obtained from the resinous exudates of Pseudognaphalium vira vira, was determined. 3beta-Hydroxy-kaurenoic acid reduced the mycelial growth of B. cinerea in solid and liquid media. Additionally, the damage produced by the fungus on the surface of tomato leaves in the presence of the diterpenoids was evaluated. A higher protective effect was observed in the presence of the hydroxylated diterpene. On the other hand, the effect of the diterpenoids on the production of enzymes that participate in the plant infection by B. cinerea was analyzed. p-Nitrophenylbutyrate esterase production was induced by both diterpenoids, whereas laccase production was only induced by the hydroxylated diterpene. In the study of the mechanism of action of these compounds, it was determined that 3beta-hydroxy-kaurenoic acid would produce permeabilization of the cell membrane of B. cinerea.