Influence of antifungal pre-treatment in preparing test mycelium on MIC values of several antifungal agents againstAspergillus niger

Antifungal activity of two imidazoles (miconazole and ketoconazole) and one polyene (amphotericin B) was evaluated using an automatic growth analysis system. Spores ofAspergillus niger were inoculated on the polylysine-coated glass bottom of a culture vessel. A colony formed in liquid medium was exposed to an antifungal agent and subsequently washed. Based on the dynamic growth rate of a test hypha selected from the colony in response to the antifungal agent, the minimum inhibitory concentration (MIC) was evaluated. The influence of time of reading (1, 2 and 3 h after washing) on the MIC determined was investigated. MICs for test hyphae subjected to antifungal pre-treatment were compared with those for hyphae without pre-treatment. Hyphae pre-treated with an antifungal agent for 1 h were found to become adapted and tolerant to that antifungal agent. Hyphae exposed and adapted to an imidazole obtained tolerance to amphotericin B as well as to the other imidazole.     

Evaluation of antifungal activity of antimycotics by automatic analyzing system

Antifungal activity of several antimycotics has been evaluated using an automatic analyzing system (AAS), which is composed of a specially designed reaction vessel, microscopic observation system, image analyzing system, and computer program for automatic tracing of hypha growth. The agar plate was prepared on the ceiling of the reaction vessel, and spore mass of a fungus (Aspergillus niger) was inoculated onto it. After the preincubation at 28 °C for 24 h the reaction vessel was set on a microscope stage and connected to the liquid flow system. An appropriate hypha was selected for the measurement of growth process during the following steps: first contact with saline for 30 min for the adaptation, the second contact with same saline for 30 min, contact with saline containing an antimycotic substance for 60 min, and contact with flushing saline for 60 min. During a sequence of these steps, the apical tip of a growing hypha displayed on a TV monitor was followed automatically. The dynamic response of hypha to an agent was analyzed by several parameters. Morphological changes of the hypha caused by respective agents were recorded on VTR for further analysis. By using this system, the antifungal activity of antimycotics could be quantitatively determined within several hours.     

Antifungal activity of a food‐grade dilution‐stable microemulsion against Aspergillus niger

Aims: To study the antifungal activities of a prepared food‐grade dilution‐stable microemulsion against Aspergillus niger. Methods and Results: Results from the antifungal activity on solid medium by agar dilution method showed that the microemulsion caused complete growth inhibition at 2000 ppm, and at 1000 ppm, showed 55% growth inhibition after 4 days of incubation and a delay of conidiation by 1 day compared with controls. Results from the antifungal activity in liquid medium by broth dilution method showed that the growth of A. niger was completely inhibited when a liquid medium containing 10⁶ spores per ml was treated with 500 ppm of microemulsion, which was determined by minimum fungicidal concentration. Study of fungicidal kinetics showed that more than 99% of viable spores were killed within 15 min. These antifungal activities were confirmed by scanning electron microscopy, light microscopy and increased Ca⁺², K⁺ and Mg⁺² leakages. Conclusions: The results suggest that the prepared microemulsions are effective antifungal systems with excellent growth inhibition and sporicidal activities, and indicate that their antifungal activity may be to the result of the disruption and dysfunction of A. niger cell walls and biological membranes. Significance and Impact of the Study: This study suggests the potential use of food‐grade dilution‐stable microemulsions for antifungal use in the food and pharmaceutical industries.     

Screening the antifungal activity of essential oils against decay fungi from palmyrah leaf handicrafts

Background

The whitish tender leaves of Palmyrah are used for making handicrafts. The problem with these articles is discolouration with time and become more brittle due to fungal attack. This could be prevented by some protective coating. Instead of expensive and harmful chemicals we decided to test natural plant essential oils to control fungal attack. Palmyrah leaf article decay fungi were isolated from two different sites of Jaffna peninsula. In this investigation Antifungal Activity of different plant essential oils from neem (Azadirachta indica), castor (Ricinus communis), citronella (Cymbopogon sp) and camphor (Cinnamomum camphora) obtained from local market have been evaluated against isolated fungi. For screening of Antifungal activity, tests and controls were set to determine minimum inhibitory concentration (MIC) and Percentage of Growth Inhibition.

Results

Morphologically three different types of Palmyrah leaf decay fungi were isolated and characterized as Aspergillus niger, Aspergillus flavus and Penicillium sp. Neem and castor oils have recorded no significant (0.05 > P) antifungal activity while citronella and camphor oils showed significantly different antifungal activity compared with control. Camphor oil and Citronella oil showed 100, 58.13% of average growth inhibition for A. niger. 96.38, 51.32% for A.flavus and 84.99, 72.76% for Penicillium sp respectively. Camphor oil showed highest percentage of growth inhibition at lowest minimum inhibitory concentration compared with citronella oil. Camphor oil was found to be highly antifungal and most effective against A niger, and A. flavus, compared with Penicillium sp and gave 100 percentage of growth inhibitions at 5, 1 and 15 ml/dl minimum inhibitory concentration respectively.

Conclusion

Significantly higher broad-spectrum of antifungal activity was observed in camphor oil than other tested oils because it showed highest percentage of growth inhibition at lowest inhibitory concentration. Therefore it could be used for the development of new environmental friendly antifungal agent for the preservation of leafy handicrafts. Further formulation, field experiments are necessary to achieve this target.     

Enhanced inhibition of Aspergillus niger on sedge (Lepironia articulata) treated with heat‐cured lime oil

Aims

This study aimed to examine heat curing effect (30–100°C) on antifungal activities of lime oil and its components (limonene, p‐cymene, β‐pinene and α‐pinene) at concentrations ranging from 100 to 300 μl ml^?1 against Aspergillus niger in microbiological medium and to optimize heat curing of lime oil for efficient mould control on sedge (Lepironia articulata).

Methods and Results

Broth dilution method was employed to determine lime oil minimum inhibitory concentration, which was at 90 μl ml^?1 with heat curing at 70°C. Limonene, a main component of lime oil, was an agent responsible for temperature dependencies of lime oil activities observed. Response surface methodology was used to construct the mathematical model describing a time period of zero mould growth on sedge as functions of heat curing temperature and lime oil concentration. Heat curing of 90 μl ml^?1 lime oil at 70°C extended a period of zero mould growth on sedge to 18 weeks under moist conditions.

Conclusions

Heat curing at 70°C best enhanced antifungal activity of lime oil against A. niger both in medium and on sedge.

Significance and Impact of the Study

Heat curing of lime oil has potential to be used to enhance the antifungal safety of sedge products.     

Automatic evaluation of antifungal volatile compounds on the basis of the dynamic growth process of a single hypha

An automatic analysing system was developed and employed for the evaluation of antifungal activity of volatile compounds in the gas phase. Aspergillus niger was inoculated on agar medium in the reaction vessel. The reaction vessel was incubated at 28° C for 24 h and then a volatile compound was introduced into the vessel either in a batch or flow manner. The antifungal activity of the respective compounds estimated in situ was expressed by the dynamic response parameters of a single hypha. All volatiles tested in the present system inhibited hyphal growth, except linalyl acetate: Limone and geraniol were the most inhibitory. In contrast, linalyl acetate promoted hyphal growth. By definition of the parameters, the fungicidal and fungistatic effects could be distinguished. Correspondence to: H. Matsuoka     

Antifungal activities of anise oil, lime oil, and tangerine oil against molds on rubberwood (Hevea brasiliensis)

The antifungal activities of anise oil, lime oil, and tangerine oil against molds identified from rubberwood surfaces (Aspergillus niger, Penicillium chrysogenum, and Penicillium sp.) were investigated. The broth dilution method was employed to determine the minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC) using the concentration of essential oils between 20 and 200 μl ml⁻¹. Inhibitory effects of the essential oils against those molds on rubberwood were also examined by means of the dip treatment and vacuum impregnation treatment. It was found that the MIC and MFC values for each treatment on agar were identical for all conditions examined. Anise oil was the strongest inhibitor with the MIC and MFC of 40 μl ml⁻¹ against Penicillium sp. and A. niger, and 60 μl ml⁻¹ against P. chrysogenum. Lime oil and tangerine oil were also effective against those molds at higher concentrations of 100–180 μl ml⁻¹. All essential oils at the MIC and MFC provided a protection from mold growth on rubberwood for at least 12 weeks at storage conditions of 30 °C with 100% RH.     

Siderophore-Producing <Emphasis Type="Italic">Alcaligenes feacalis</Emphasis> Exhibited More Biocontrol Potential Vis-à-Vis Chemical Fungicide

We report the in vitro phytopathogen suppression activity of siderophoregenic preparations of Alcaligenes feacalis vis-à-vis the oraganochlorine fungicide, bavistin. Siderophore-rich culture broth, siderophore-rich supernatant, and purified siderophore preparation exerted antifungal activity against Aspergillus niger NCIM 1025, A. flavus NCIM 650, Fusarium oxysporum NCIM 1008, and Alternaria alternata IARI 715. Among all the preparations, siderophore-rich broth exhibited potent antifungal activity. The minimum fungicidal concentration required was 75 μl for A. niger and F. oxysporum and 50 μl for A. flavus and A. alternata.     

Antibacterial and antifungal activity of ethanolic extracts from eleven spice plants

Eleven ethanolic extracts from spices of Melissa officinalis, Mentha piperita, Laurus nobilis, Rhus coriaria, Dianthus coryophyllum, Piper nigrum, Capsicum annum, Juniperus oxycedrus, Erica arborea, Colutea arborescens, and Cuminum cyminum collected from various regions of Turkey and local markets were assayed for the in vitro antibacterial activity against 3 Gram-positive (Bacillus subtilis, Staphylococcus aureus and S. epidermidis) and 2 Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa), using agar dilution methods. In addition, their possible toxicity to Candida albicans and Aspergillus niger was determined, using both agar dilution and disc-diffusion methods. The minimum inhibition concentration (MIC) of the M. piperita, L. nobilis and J. oxycedrus ethanolic extracts was 5 mg/mL for all the microorganisms tested. P. aeruginosa was the most sensitive bacterial strain to P. nigrum and E. arborea extracts among both Gram-positive and Gram-negative bacteria tested with MIC of 5 mg/mL. The extracts of L. nobilis, D. coryophyllum, J. oxycedrus and C. arborescens showed higher inhibitory activity against the yeast C. albicans and the fungus A. niger than the standard antifungal nystatin.     

Toxicity of Some Terpenoids Against Fungi Infesting Fruits and Seeds of Capsicum annuum L. During Storage

On mycoflora analysis of dried fruits and seeds of chilli stored for six months, Aspergillus flavus and A. niger were the more frequent out of eighteen fungi isolated. Some terpenoids viz, Citral, Citronellal, Citronellol, Eugenol, Farnesol and Nerol showed absolute toxicity against A. flavus and A. niger at 0.5 per cent concentration. The minimum inhibitory concentration (MIC) and nature (fungistatic/fungicidal) of all the terpenoids against both these fungi were determined, Citral, Eugenol and Nerol exhibited a broad range of toxicity and were found to be more efficacious than some synthetic fungicides.     

Optimization of process parameters for improved production of bioactive metabolite by a novel endophytic fungus <Emphasis Type="Italic">Fusarium</Emphasis> sp. DF2 isolated from <Emphasis Type="Italic">Taxus wallichiana</Emphasis> of North East India

An endophytic fungus having antifungal and antibacterial properties was isolated from Taxus wallichiana of Arunachal Pradesh, India. On the basis of morphological and molecular characteristics, the fungus was identified as Fusarium sp. and designated as DF2. The fungus was optimized for growth and maximum production of the antimicrobial agent. Media with 5% leaf extract (w/v) supplemented with 0.1% dextrose as carbon and yeast extract as nitrogen source favored the growth with temperature optimum 25 ± 2°C and pH 6. Incubation period of 10 days was observed optimum for growth and production of antimicrobial agent. Phenylalanine and dextrose enriched basal medium promoted the antimicrobial agent production, whereas methionine amended in combination with glucose promoted higher biomass accumulation. The TLC purified active compound with UV λ-max 270 nm in ethyl acetate has got the lowest minimum inhibitory concentration (MIC) against Bacillus subtilis, Staphylococcus aureus and Escherichia coli and highest against Pseudomonas aeruginosa.     

Synthesis and in vitro microbiological evaluation of novel diethyl 6,6′-(1,4-phenylene)bis(4-aryl-2-oxo-cyclohex-3-enecarboxylates)

Novel bis cyclohexenone ester derivatives 14–19 were synthesized and characterized by their spectral data. In vitro microbiological evaluations were carried out for all the novel compounds 14–19 against clinically isolated bacterial and fungal strains. Compounds 15, 16, 18 against Staphylococcus aureus, 14, 15 against β-Haemolytic streptococcus, 15, 19 against Micrococcus luteus, 17, 18 against Salmonella typhii, 14, 17 against Shigella flexneri, 15 against Escherichia coli, 16 against Pseudomonas aeruginosa, 15, 18, 19 against Klebsiella pneumonia exhibited potent antibacterial activity at an minimum inhibitory concentration (MIC) value of 6.25 μg/ml, whereas compound 16 against Aspergillus flavus, 17 against A. niger, 16, 18 against Mucor indicus, 15, 17–19 against Microsporum gypseum revealed excellent antifungal activity at an MIC value of 6.25 μg/ml.     

Antimycotic Activity Potentiation of Allium sativum Extract and Silver Nanoparticles against Trichophyton rubrum

A natural and biocompatible extract of garlic as a support, decorated with silver nanoparticles, is a proposal to generate an effective antifungal agent against dermatophytes at low concentrations. Silver nanoparticles (AgNPs) with a diameter of 26±7 nm were synthesized and their antimycotic activity was examined against Trichophyton rubrum (T. rubrum), inhibiting 94 % of growth at a concentration of 0.08 mg ml^?1. Allium sativum (garlic) extract was also obtained (AsExt), and its MIC was 0.04 mg ml^?1. To increase the antifungal capacity of those systems, AsExt was decorated with AgNPs, obtaining AsExt‐AgNPs. Using an AsExt concentration of 0.04 mg ml^?1 in independent experiments with concentrations from 0.01 to 0.08 mg ml^?1 of AgNPs, it was possible to inhibit T. rubrum at all AgNPs concentrations; it proves a synergistic effect between AgNPs and AsExt. Even if 1 % of the minimum inhibitory concentration of AsExt (0.0004 mg ml^?1) is used, it was possible to inhibit T. rubrum at all concentrations of AgNPs, demonstrating the successful antimycotic activity potentiation when combining AsExt and AgNPs.     

In vitro activity of 15-azasterol (A25822B) against chalkbrood pathogen Ascosphaera apis in the honey bee

The antifungal agent 15-azasterol A25822B was examined for effects on the growth and development of Ascosphaera apis. The minimum inhibition concentration (MIC) of azasterol against A. apis was 1 m. Growth and development of A. apis was completely controlled at this concentration. At a concentration of 0.01 m growth of A. apis was retarded and although sporocysts were formed developing spores were not be able to reach maturation. A major effect of azasterol at this low concentration was the accumulation of lipid in the hyphae, sporocysts and immature spores. In addition it caused a conformational change in mitochondria and damage to the spore membrane structure. On the basis of these results, further investigations of azasterol for the treatment of chalkbrood disease in the honey bee are warranted.Work was performed during sabbatical leave at the University of California, Davis.     

Biotoxic effects of Sumisclex and Carbomar fungicides on four Aspergillus species isolated from the rhizosphere of some Taif plants

Abstract

The antifungal activities of two systemic fungicides (Sumisclex and Carbomar) on four rhizospheric Aspergillus species (A. candidus, A. sydawi, A. niger1 and A. niger2) were studied. Also the phytotoxicity against some saudian seeds was analysed. Results indicated that Sumisclex fungicide was more effective on the growth of A. candidus and A. sydawi under laboratory conditions (the IC₅₀ of 3 and 4 ppm were recorded, respectively) than Carbomar where an IC₅₀ of 11 ppm for A. candidus and 35 ppm for A. sydawi were observed. The growth of the two strains of A. niger was completely inhibited by Carbomar even at the lowest 10 ppm concentration. On the other hand results revealed that phytotoxicity was apparently linked with the type of the fungicide and its concentration. However, the root system was more sensitive to the tested fungicides especially at higher concentrations.     

Standardization of hyphal growth inhibition rate as a means of evaluating Microsporum spp. in vitro susceptibility to terbinafine, griseofulvin, and ciclopiroxolamine

Reference methods for antifungal susceptibility tests recommend the use of conidia as inoculum. However, some isolates produce few conidia, while the invasive form of filamentous fungi in general is hyphae making susceptibility tests infeaseble. These facts suggest that other than conidia broth dilution method is required for susceptibility tests. The aim of this study was to clarify if the hyphal growth inhibition rate could be used as a method of determining the antifungal susceptibility of genus Microsporum. For this reason, a method which traces hyphal tips automatically and measures their growth rate was standardized for Microsporum spp. Control growth curves and test growth curves obtained by real-time observation of the hyphae groups responses to different concentrations of terbinafine, griseofulvin, and ciclopiroxolamine were used to compare with minimum inhibitory concentrations (MICs) obtained by conidia broth microdilution method. A visible reduction in the growth inhibition rate was observed when hyphal activity was evaluated using the third or fourth serial two-fold dilution below the MIC determined by broth microdilution for terbinafine and ciclopiroxolamine. For griseofulvin, this reduction occurred after the fifth dilution below the MIC. This study highlights the importance of the inoculum type used to determine the in vitro susceptibility of Microsporum strains. We conclude that measurement of hyphal growth inhibition, despite being time consuming, could be a suitable method for evaluating antifungal susceptibility, particularly for fungi as Microsporum spp. that produce a small (or not at all) number of conidia.     

Cooperative Anti-Candida Effects of Lactoferrin or Its Peptides in Combination with Azole Antifungal Agents

The effects of lactoferrin (LF), an antimicrobial protein secreted in body fluids, and its peptides in combination with azole antifungal agents were investigated by the micro-broth-dilution method in a study of Candida albicans. In the case of LF, its pepsin hydrolysate (LFhyd) or the LF-derived antimicrobial peptide Lactoferricin® B (LF-B), the concentrations required to inhibit the growth of Candida decreased in the presence of relatively low concentrations of clotrimazole (CTZ). The minimum inhibitory concentration (MIC) of all azole antifungal agents tested was reduced by 1/41/16 in the presence of a sub-MIC level of each of these LF-related substances. Polyene and fluoropyrimidine antifungal agents did not show such a combined effect with these LF-related substances. The anti-Candida activity of LF or LF-B in combination with CTZ was shown to be synergistic by checkerboard analysis. These results indicate that LF-related substances function cooperatively with azole antifungal agents against C. albicans.     

Transmission of the eect of an antifungal agent within a single hypha

A micro-compartment culture method was devised in which a single hypha of Rhizopus stolonifer growing on an agar section traversed an antifungal non-diffusible barrier to another agar section; thus the local environment of the distal or proximal part of the hypha could be controlled independently. The responses in terms of hyphal extension of the test fungus to local application of amphotericin B in this culture system were estimated by using an automatic analysing system. After hyphae had traversed the barrier, distal application of amphotericin B caused no appreciable effect on the proximal hyphae. In contrast, proximal application of amphotericin B caused inhibition of the extension of distal hyphae. The reversal of polarized cytoplasmic streaming also occured during the inhibition of distal hyphal extension. The extents of inhibition of the distal hyphal extension and the cytoplasmic streaming were dependent upon the hyphal distance between the amphotericin B application site and the hyphal tip. These results show that the effect of an antifungal agent on a hypha depends on the region of the hypha exposed. Cytoplasmic streaming may play key role in the transmission of antifungal effects within a single hypha.     

Antifungal activities of essential oils applied by dip-treatment on areca palm (Areca catechu) leaf sheath and persistence of their potency upon storage

The antifungal activities of cinnamon oil, clove oil, anise oil, and peppermint oil, and their main components (cinnamaldehyde, eugenol, trans-anethole, and menthol, respectively) against molds identified from areca palm leaf sheath (Mucor dimorphosporus, Penicillium sp., Aspergillus niger, and Rhizopus sp.) were investigated. An agar dilution method was employed to determine the minimum inhibitory concentration (MIC) of essential oils and their main components. Zone inhibition tests and the inhibitory effect of the leaf sheath dip-treated with essential oils against those molds were examined. Major components of essential oils on the leaf sheath during storage were quantified by gas chromatography analysis. The MIC values of essential oils on agar and on the leaf sheath were identical. With an MIC of 50 ??g ml⁻¹, cinnamon oil had the strongest inhibitory effect. At their MICs the oils were capable of providing protection against mold growth on the leaf sheath for at least 12 weeks during storage at 25 °C and 100% RH. Scanning electron microscope examination showed that essential oils prevented spore germination. Except for menthol in peppermint oil, the main components of the essential oils, which were fairly stable over the storage period, largely contributed to the antifungal activity.     

响应面法优化黑曲霉固定化条件及其对土壤中溴氰菊酯的降解特性研究

【目的】通过研究吸附包埋法固定黑曲霉(Aspergillus niger)的最佳制备工艺,初步探讨固定化黑曲霉对溴氰菊酯(deltamethrin, DM)及其中间产物3-苯氧基苯甲酸(3-phenoxybenzoic acid,3-PBA)的降解机制,并将其应用于农业种植中以评价固定化黑曲霉的实际应用效果。【方法】以生物炭、海藻酸钠为固定化载体,通过单因素和响应面试验对固定化黑曲霉(immobilized Aspergillusniger)的制备工艺进行优化。同时,利用高效液相色谱法分析DM和3-PBA的含量变化。【结果】海藻酸钠浓度、生物炭浓度和菌液接种量为DM去除率的显著影响因子,当三者分别为25.27、1.28和125.28 g/L时,是黑曲霉固定化的最佳制备条件;在施加固定化黑曲霉后,土壤中DM半衰期由7.6d缩短至5.2d,黑曲霉对3-PBA也具有降解作用,在21h达到最低浓度1.45mg/kg;修复后的土壤可显著提高番茄种子发芽率,株高、根长等6个生长指标较DM单独处理组也有不同程度的恢复;在经固定化黑曲霉修复28 d后,污染土壤根系酶活和微生物数量均得到不同程度改善。【...