Synthesis and antifungal activities of novel thiophene-based stilbene derivatives bearing an 1,3,4-oxadiazole unit.

BACKGROUND: Natural stilbenes (especially resveratrol and its derivatives) are well-known phytoalexins that are active against many plant diseases. However, oxidative degradation and low bioavailability limit their exogenous application as fungicides on crops. In this study, a new class of resveratrol-inspired thiophene-based stilbene derivatives bearing an 1,3,4-oxadiazole unit was synthesized and the derivatives' antifungal activities against phytopathogenic fungi were investigated. RESULTS: The results revealed that compounds 5h and 5j exhibited improved antifungal activity against Botrytis cinerea with median effective concentrations (EC50 ) of 168.5 and 155.4 µg mL-1 , respectively, which were superior to the EC50 of resveratrol (263.1 µg mL-1 ). Compound 5j was shown to effectively control disease development in B. cinerea-infected tomatoes in vivo. Notably, considerably abnormal mycelial morphology and increased cell membrane conductivity were observed in the presence of compound 5j. CONCLUSION: A new class of thiophene-containing stilbene derivatives was designed and synthesized. Bioassay results showed that compound 5j exhibited promising antifungal activity, suggesting practical potential for fungal disease control. © 2018 Society of Chemical Industry.

Evaluation of cytotoxicity, genotoxicity and ecotoxicity of nanoemulsions containing Mancozeb and Eugenol.

Mancozeb is a fungicide widely used in agriculture, mostly against the pathogen Glomerella cingulata responsible for the rot of ripe grape, but presents high toxicity. Strategies are sought to reduce the toxicity of this fungicide and alternative treatments are welcome. An alternative could be the use of clove oil, which has Eugenol as its major compound, and has antifungal potential against G. cingulata, however, Eugenol is susceptible to degradation processes which may compromise its efficacy. The nanoencapsulation of Mancozeb and Eugenol is a possible strategy to overcome the limitations of toxicity, solubility and instability of these compounds. Therefore, the objective of this study is to develop nanoemulsions containing Mancozeb (0.1 mg/mL) and Eugenol (33 mg/mL), isolated or associated, and evaluate the safety of these formulations through cytotoxicity, genotoxicity and ecotoxicity tests. Nanoemulsions were developed by the spontaneous emulsification method, cytotoxicity and genotoxicity were evaluated in healthy human cells through MTT, Dichlorofluorescein diacetate and Picogreen tests, and ecotoxicity assessment was carried out using the chronic toxicity test in springtails. After preparation, the physicochemical characterization of the nanoemulsions were performed which presented mean particle size between 200 and 300 nm, polydispersity index less than 0.3, negative zeta potential and acid pH. The nanoencapsulation was able to avoid the reduction of the cell viability caused by Mancozeb, while Eugenol was shown to be safe for cell use in both free and nanostructured forms, however the association of the two active compounds showed toxicity in the higher doses of Mancozeb. In the ecotoxicity tests, both free Mancozeb and Eugenol forms presented high toxic potential for soil, whereas the nanoencapsulation of these compounds did not cause a reduction in number of springtails. Therefore, from the tests performed, it was possible to observe that nanoencapsulation of Mancozeb and Eugenol is a safe alternative for the application of these compounds mainly in agriculture.

Synthesis and Antifungal Activities of Drimane-Amide Derivatives from Sclareol.

AIM AND OBJECTIVE: Plant diseases are caused by fungal pathogens lead to severe economic losses in many agriculture crops. And the increasing resistance of many fungi to commonly used antifungal agents necessitates the discovery and development of new fungicides. So this study was focused on synthesizing novel skeleton compounds to effectively control plant diseases. MATERIALS AND METHODS: A series of drimane-amide derivatives were designed, synthesized by aminolysis reaction of amine with intermediate sclareolide which was prepared from sclareol. The structures of all the synthesized compounds were confirmed using 1H NMR, 13C NMR, and HRMS (ESI) spectroscopic data. Their in vitro antifungal activity were preliminarily evaluated by using the mycelium growth rate method against five phytopathogenic fungi: Botrytis cinerea, Glomerella cingulata, Alternaria alternate, Alternaria brassicae, and Fusarium graminearum. RESULTS: 23 target compounds were successfully obtained in yields of 52-95%. Compounds A2 and A3 displayed favorable inhibitory potency against B. cinerea, G. cingulata and A. brassicae with IC50 values ranging from 3.18 to 10.48 µg/mL. These two compounds displayed higher fungicidal activity than sclareol against all the tested phytopathogenic fungi, and were more effective than the positive control thiabendazole against A. alternate and A. brassicae. The structure-activity relationship studies of compounds A1-10 indicated that both the position and type of substituent on the phenyl ring had significant effects on antifungal activity. CONCLUSION: The drimane-amide derivatives A2 and A3 were the most promising derivatives and should be selected as new templates for the potential antifungal agents.

Synthesis, characterization and antifungal efficacy of C-coordinated O-carboxymethyl chitosan Cu(II) complexes.

A novel type of O-carboxymethyl chitosan Schiff bases (O-CSPX) was synthesized via a condensation reaction. After the coordination reaction of cupric ions, Cu(II) complexes (O-CSPX-Cu) were achieved. The theoretical structure of O-CSPX-Cu calculated by Gaussian 09 reveals that the copper ions underwent dsp2 hybridization, coordinated by the carbon atom in the p-π conjugate group and the oxygen atoms in the acetate ion. Then, the structures were confirmed by FT-IR, 1H NMR, CP-MAS 13C NMR, elemental analysis, DSC and XRD. The antifungal properties of O-CSPX-Cu against Phytophthora capsici (P. capsici), Gibberella zeae (G. zeae) and Glomerella cingulata (G. cingulata) were evaluated at concentrations ranging from 0.05mg/mL to 0.20mg/mL. The experiments indicated that the derivatives have significantly enhanced antifungal activity after copper ion complexation compared with the original chitosan. Moreover, it was shown that 0.20mg/mL of O-CSP3-Cu and O-CSP4-Cu can 100% inhibit the growth of P. capsici. The experimental results reveal that the antifungal efficiency is related to the space steric hindrance on the benzene ring, which may provide a novel direction for the development of copper fungicides.

Favorable Outcomes of Filamentous Fungal Endophthalmitis Following Aggressive Management.

PURPOSE: To evaluate the visual outcomes following aggressive management of filamentous fungal endophthalmitis with prompt surgical intervention and oral and intravitreal voriconazole. METHODS: Retrospective chart review study of consecutive patients with culture- or biopsy-proven filamentous fungal endophthalmitis treated at an academic referral center. Clinical characteristics, treatment regimens, and visual outcomes were analyzed. RESULTS: Included were 5 patients, 1 with endogenous endophthalmitis due to systemic fusariosis and 4 due to exogenous endophthalmitis (1 with Fusarium, 2 with Scedosporium apiospermum, and 1 with Glomerella spp.). On presentation, 1 patient had best-corrected visual acuity (BCVA) of 20/20. The remaining 4 patients had count-fingers to hand motion (HM) vision. All patients underwent immediate surgical intervention for infection control. All patients received oral or intravenous voriconazole and aggressive intravitreal voriconazole every 2-3 days initially. Intravitreal amphotericin was added if there was poor response to voriconazole alone. Three patients achieved a final BCVA of 20/20, 1 patient achieved BCVA of 20/50, and 1 remained HMs only. CONCLUSION: Aggressive treatment of filamentous fungal endophthalmitis with early surgical intervention, systemic antifungal therapy, and frequent intravitreal injections of voriconazole can result in excellent visual outcomes in some patients.

Salicylic acid confers enhanced resistance to Glomerella leaf spot in apple.

Glomerella leaf spot (GLS) caused by Glomerella cingulata is a newly emergent disease that results in severe defoliation and fruit spots in apple. Currently, there are no effective means to control this disease except for the traditional fungicide sprays. Induced resistance by elicitors against pathogens infection is a widely accepted eco-friendly strategy. In the present study, we investigated whether exogenous application of salicylic acid (SA) could improve resistance to GLS in a highly susceptible apple cultivar (Malus domestica Borkh. cv. 'Gala') and the underlying mechanisms. The results showed that pretreatment with SA, at 0.1-1.0 mM, induced strong resistance against GLS in 'Gala' apple leaves, with SA treated leaves showing significant reduction in lesion numbers and disease index. Concurrent with the enhanced disease resistance, SA treatment markedly increased the total antioxidant capacity (T-AOC) and defence-related enzyme activities, including catalase (CAT), superoxide dismutase (SOD), peroxidase (POD), phenylalanine ammonia-lyase (PAL) and polyphenol oxidase (PPO). As expected, SA treatment also induced the expression levels of five pathogenesis-related (PR) genes including PR1, PR5, PR8, Chitinase and ß-1,3-glucanase. Furthermore, the most pronounced and/or rapid increase was observed in leaves treated with SA and subsequently inoculated with G. cingulata compared to the treatment with SA or inoculation with the pathogen. Together, these results suggest that exogenous SA triggered increase in reactive oxygen species levels and the antioxidant system might be responsible for enhanced resistance against G. cingulata in 'Gala' apple leaves.

Synthesis of heterocycle-attached methylidenebenzenesulfonohydrazones as antifungal agents.

A series of heterocycle-attached methylidenebenzenesulfonohydrazone derivatives were synthesized and evaluated for their antifungal activities against seven phytopathogenic fungi such as Fusarium graminearum, Alternaria solani, Valsa mali, Phytophthora capsici, Fusarium solani, Botrytis cinerea, and Glomerella cingulata. Compounds 7b, 8d, 9a, 9b and 9d exhibited a good and broad-spectrum of antifungal activities against at least five phytopathogenic fungi at the concentration of 100 µg/mL. It demonstrated that addition of one double bond between the phenylsulfonylhydrazone fragment and the furan ring of 6a,b,d could afford more active compounds 9a,b,d; however, introduction of the nitro group on the phenyl ring of 6a-9a gave less potent compounds 6e-9e.

Identification and Bioactivity of Compounds from the Mangrove Endophytic Fungus Alternaria sp.

Racemic new cyclohexenone and cyclopentenone derivatives, (±)-(4R*,5S*,6S*)-3-amino-4,5,6-trihydroxy-2-methoxy-5-methyl-2-cyclohexen-1-one (1) and (±)-(4S*,5S*)-2,4,5-trihydroxy-3-methoxy-4-methoxycarbonyl-5-methyl-2-cyclopenten-1-one (2), and two new xanthone derivatives 4-chloro-1,5-dihydroxy-3-hydroxymethyl-6-methoxycarbonyl-xanthen-9-one (3) and 2,8-dimethoxy-1,6-dimethoxycarbonyl-xanthen-9-one (4), along with one known compound, fischexanthone (5), were isolated from the culture of the mangrove endophytic fungus Alternaria sp. R6. The structures of these compounds were elucidated by analysis of their MS (Mass), one and two dimensional NMR (nuclear magnetic resonance) spectroscopic data. Compounds 1 and 2 exhibited potent ABTS [2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)] scavenging activities with EC50 values of 8.19 ± 0.15 and 16.09 ± 0.01 µM, respectively. In comparison to Triadimefon, compounds 2 and 3 exhibited inhibitory activities against Fusarium graminearum with minimal inhibitory concentration (MIC) values of 215.52 and 107.14 µM, respectively, and compound 3 exhibited antifungal activity against Calletotrichum musae with MIC value of 214.29 µM.

Synthesis and antifungal activity of aromatic bis-gamma-lactones analogous to avenaciolide.

Avenaciolide is a bis-gamma-lactone isolated from Aspergillus avenaceus and possesses antifungal activity. Here, we describe the total syntheses and characterization by elemental analyses, and IR and NMR spectroscopy of three new bis-gamma-lactones analogous to avenaciolide, where the octyl group of the natural product was replaced by aromatic groups. The effects of the avenaciolide, the novel compounds, and their synthetic precursors on the mycelia development and conidia germination of Colletotrichum gloeosporioides were evaluated in vitro. The new compounds were as active as avenaciolide in the tested conditions, while the synthetic precursors were inactive. The preparation and characterization of 15 new synthetic intermediates are also described.

Dammarane triterpenoids from the roots of Gentiana rigescens.

Six new minor dammarane triterpenoids were isolated from the roots of Gentiana rigescens. These include one aglycone, gentirigenic acid (1), and five glycosides, gentirigeosides A-E (2-6). Their structures were elucidated through detailed spectroscopic analyses, including 1D and 2D NMR techniques, and enzymatic hydrolysis. Antifungal testing of these compounds showed that the glycosides gentirigeosides A (2), C (4), and E (6) had antifungal activity against the plant pathogen Glomerella cingulata.

Inhibition of plant-pathogenic fungi by the barley cystatin Hv-CPI (gene Icy) is not associated with its cysteine-proteinase inhibitory properties.

The recombinant barley cystatin Hv-CPI inhibited the growth of three phytopathogenic fungi (Botrytis cinerea, Colletotrichum graminicola, and Plectosphaerella cucumerina) and the saprotrophic fungus Trichoderma viride. Several mutants of barley cystatin were generated by polymerase chain reaction approaches and both their antifungal and their cysteine-proteinase inhibitory properties investigated. Point mutants R38-->G, Q63-->L, and Q63-->P diminished their capacity for inhibiting papain and cathepsin B, retaining their antifungal properties. However, mutant C68-->G was more active for papain and cathepsin B than the wild type. These results indicate that in addition to the consensus cystatin-reactive site, Q63-V64-V65-A66-G67, the A37-R38-F39-A40-V41 region, common to all cereal cystatins, and the C68 residue are important for barley cystatin activity. On the other hand, the K92-->P mutant is inactive as a fungicide, but still retains measurable inhibitory activity for papain and cathepsin B. Against B. cinerea, the antifungal effect of Hv-CPI and of its derived mutants does not always correlate with their activities as proteinase inhibitors, because the Q63-->P mutant is inactive as a cystatin, while still inhibiting fungal growth, and the K92-->P mutant shows the reciprocal effects. These data indicate that inhibition of plant-pathogenic fungi by barley cystatin is not associated with its cysteine-proteinase inhibitory activity. Moreover, these results are corroborated by the absence of inhibition of intra- and extramycelia-proteinase activities by barley cystatin and by other well-known inhibitors of cysteine-proteinase activity in the fungal zymograms of B. cinerea.

Extracts from seaweeds can promote fungal growth.

Hormesis is the stimulation of a biological response at low concentrations of an inhibitor. Ethanolic extracts were made using Osmundaria serrata (Suhr) R. E. Norris and Stypopodium zonale (Lamouroux) Papenfuss from the East coast of South Africa. Two plant pathogens (Colletotrichum gloeosporioides (Penz.) Penz. and Sacc. and Rhizoctonia solani Kühn) were used as test organisms in bioassays. Serial dilutions of macroalgal extracts were tested by the pour plate technique. Both growth inhibitory and promotory responses were observed. The hormetic response was observed in both the fungi when grown on low dilutions of ethanol and the O. serrata extract, and when R. solani was grown on the S. zonale extract. This study provides more evidence of hormesis in macroalgal products and the phenomenon is discussed in relation to its possible cause and significance in the application of seaweed extracts.

Peptide inhibitors of appressorium development in Glomerella cingulata.

The phytopathogen Glomerella cingulata (anamorph: Colletotrichum gloeosporioides) infects host tissue by means of a specialised infection structure, the appressorium. The Saccharomyces cerevisiae alpha-mating factor pheromone, the Saccharomyces kluyveri alpha-mating factor pheromone and a hendecapeptide produced by G. cingulata inhibit appressorium development. The amino acid sequence of the G. cingulata peptide, GYFSYPHGNLF, is different from that of the mature pheromone peptides of other filamentous fungi. The peptide has sequence similarity with the Saccharomyces alpha-mating factor pheromones, but is unable to elicit growth arrest in S. cerevisiae.