Voriconazole resistance genes in Aspergillus flavus clinical isolates.

OBJECTIVE: The present study was designed to discover novel biomarkers involved in voriconazole resistance in clinical isolates of Aspergillus flavus. MATERIALS AND METHODS: Two voriconazole non-wild-type and two voriconazole-wild-type A. flavus clinical isolates were selected to evaluate possible molecular mechanism involved in A. flavus resistance to voriconazole using the mutation assessment, Quantitative real- time PCR of cyp51A and cyp51C genes and complementary DNA- amplified fragment length polymorphism technique. RESULTS: No mutations were seen in the cyp51A and cyp51C genes in voriconazole non-wild-type isolates compared to wild- type and reference strains. Regarding to mRNA expression results, no changes were observed in expression fold of cyp51A and cyp51C mRNA expression level in first non- wild- type isolate compared to wild-type isolate. For second isolate cyp51C mRNA expression level was down regulated (5.6 fold). The set of genes including ABC fatty acid transporter XM- 002375835 and aldehydereductase XM- 002376518 and three unknown functional genes were identified. Based on results, the over-expression of AKR1 and ABC fatty acid transporter in the voriconazole non- wild- type isolates suggests these genes could represent a novel molecular marker linked to the voriconazole resistance in A. flavus. CONCLUSION: The results obtained in this study showed a novel finding as the authors identified AKR1 and ABC fatty acid transporter genes as possible voriconazole target genes in Iranian clinical isolates of A. flavus.

Antimicrobial activity of green silver nanoparticles from endophytic fungi isolated from Calotropis procera (Ait) latex.

Endophytes, a potential source of bioactive secondary metabolites, were isolated from the widely used medicinal plant Calotropis procera Ait. Approximately 675 segments from 15 Calotropis procera plants and 15 latex samples were assessed for the presence of endophytic fungi. Finally, eight fungal species were isolated and identified based on their macro- and micro-morphology. The endophytic fungal filtrates were screened for their antimicrobial activity against 11 important pathogenic micro-organisms. The filtrates of nanoparticles were from three of the eight isolated endophytic fungi, namely, Penicillium chrysogenum, Aspergillus fumigatus and Aspergillus flavus, and were highly effective against the tested bacteria, while the remaining endophytic fungal filtrates displayed low activity.

Cyclopiazonic Acid Is a Pathogenicity Factor for Aspergillus flavus and a Promising Target for Screening Germplasm for Ear Rot Resistance.

Aspergillus flavus, an opportunistic pathogen, contaminates maize and other key crops with carcinogenic aflatoxins (AFs). Besides AFs, A. flavus makes many more secondary metabolites (SMs) whose toxicity in insects or vertebrates has been studied. However, the role of SMs in the invasion of plant hosts by A. flavus remains to be investigated. Cyclopiazonic acid (CPA), a neurotoxic SM made by A. flavus, is a nanomolar inhibitor of endoplasmic reticulum calcium ATPases (ECAs) and a potent inducer of cell death in plants. We hypothesized that CPA, by virtue of its cytotoxicity, may serve as a key pathogenicity factor that kills plant cells and supports the saprophytic life style of the fungus while compromising the host defense response. This proposal was tested by two complementary approaches. A comparison of CPA levels among A. flavus isolates indicated that CPA may be a determinant of niche adaptation, i.e., isolates that colonize maize make more CPA than those restricted only to the soil. Further, mutants in the CPA biosynthetic pathway are less virulent in causing ear rot than their wild-type parent in field inoculation assays. Additionally, genes encoding ECAs are expressed in developing maize seeds and are induced by A. flavus infection. Building on these results, we developed a seedling assay in which maize roots were exposed to CPA, and cell death was measured as Evans Blue uptake. Among >40 maize inbreds screened for CPA tolerance, inbreds with proven susceptibility to ear rot were also highly CPA sensitive. The publicly available data on resistance to silk colonization or AF contamination for many of the lines was also broadly correlated with their CPA sensitivity. In summary, our studies show that i) CPA serves as a key pathogenicity factor that enables the saprophytic life style of A. flavus and ii) maize inbreds are diverse in their tolerance to CPA. Taking advantage of this natural variation, we are currently pursuing both genome-wide and candidate gene approaches to identify novel components of maize resistance to Aspergillus ear rot.

Hongos productores de micotoxinas asociados a hierbas medicinales en la Ciudad de São Paulo, Brasil / Mould and mycotoxins associated in medicinal plants in São Paulo, Brazil

Antecedentes: la fitoterapia es una de las más antiguas prácticas utilizadas por la humanidad. Hasta mediados del siglo XIX, cuando se introdujeron los medicamentos, la formulación de estos generalmente era basada en plantas medicinales. Objetivos: Determinar la micobiota y los niveles de aflatoxinas originadas de Aspergillus sección Flavi aislados de las 50 muestras de medicamentos fitoterápicos comercializados actualmente en la ciudad de São Paulo, Brasil. Métodos: Cincuenta (50) muestras de medicamentos fitoterápicos en la forma de hojas (té-25) y cápsulas (25) fueron colectadas de agosto de 2000 a julio de 2001. Los hongos filamentosos aislados fueron identificados al nivel de género de acuerdo con las características morfológicas y criterios taxonómicos. El análisis de aflatoxinas fue realizada por cromatografía de capa fina (TLC). Resultados: El análisis microbiológico mostró que 41 (82 por ciento) de los medicamentos fitoterápicos presentaron un crecimiento fúngico sobre las 100 UFC/g. Un total de 106 especies de seis diferentes géneros fueron aislados (Aspergillus, Penicillium, Mucor, Rhizopus y Alternaria). El género Aspergillus fue el predominante (60.5 por ciento) seguido por Penicillium (20,0 por ciento). Aspergillus niger (30 por ciento) A. flavus (22 por ciento), A. fumigatus (6,5 por ciento) y A. parasiticus fueron las especies de Aspergillus identificadas. Se observó que 13 (56,5 por ciento), de los 23 A. flavus aislados y dos aislados de A. parasiticus produjeron aflatoxinas. Conclusiones: La contaminación observada en la mayoría de los productos y el alto nivel de cepas productoras de aflatoxinas justifica un análisis más cuidadoso de los medicamentos fitoterápicos comercializados y la aplicación de leyes más rigurosas son necesarias para garantizar la calidad de los productos.

Background: phytotherapy is one of the most ancient practices used by humanity. In Antiquity until the middle of the XIX century, when chemotherapeutic drugs were introduced, formulation of medicines was usually based on medicinal plants. Objective: To determine mycobiota and levels of Aspergillus section Flavi aflatoxins isolated from 50 samples of phytotherapeutic remedies currently commercialized in São Paulo, Brazil. Methods: Fifty (50) samples of phytotherapeutic remedies in the form of leaves (teas-25) and powders (capsules-25) were collected from August 2000 to July 2001. Filamentous fungi isolates were identified at the genera level in accordance with morphological characteristics and taxonomic criteria. Aflatoxins were performed by Thin-layer chromatography (TLC). Results: The microbiological analysis showed that 41 (82 percent) of phytotherapeutic remedies presented a fungal growth over 100 CFU/g. A total of 106 species of six different genera were isolated (Aspergillus, Penicillium, Mucor, Rhizopus and Alternaria). The genus Aspergillus was the predominant (60.5 percent) followed by Penicillium genus (20.0 percent). Aspergillus niger (30 percent) A. flavus (22 percent), A. fumigatus (6.5 percent) and A. parasiticus were the species of Aspergillus identified. It was observed that 13 (56.5 percent) of 23 A. flavus isolates and two A. parasiticus isolates produced aflatoxins. Conclusions: The contamination observed in most products and the high level of aflatoxigenic strains justify the concern regarding the execution of more careful analyzes of the commercialized phytotherapeutic remedies and the application of more rigorous laws that may warrant the quality of these products.

MYCOLOGICAL ANALYSIS AND AFLATOXIN B1 CONTAMINANT ESTIMATION OF HERBAL DRUG RAW MATERIALS.

BACKGROUND: The present study explores the fungal contamination of important herbal drug raw materials (HDRM), which are widely used in the preparation of many herbal drugs. Understanding of the microbial contamination status of HDRM is one of the important steps to ensure the safety and efficacy of herbal drugs. MATERIALS AND METHODS: Eighteen samples of six herbal drug raw materials (HDRM) viz., Acorus calamus Linn., Cassia angustifolia Vahl., Centella asiatica (Linn.) Urban, Myristica fragrans Houtt., Tinospora cardifolia (Wild) Miers and Withania somnifera (Linn.) Dunal, were screened for fungal contamination, by employing serial dilution method. All the isolates of Aspergillus flavus were screened for their ability to produce aflatoxin B1 (AB1) and highly contaminated samples were subjected to AB1 estimation by using Thin Layer Chromatography (TLC), spectrophotometric method and occurrence of Aflatoxin B1 was confirmed by Liquid Chromatography-Mass Spectrometry analysis (LCMS). RESULTS: A total of 302 isolates of 42 fungal species belonging to 17 genera were found in association with test the samples. More than 61% of A. flavus isolates tested positive for production of AB1 and highest yield recorded was 5008.20 ppb from the isolates of T. cordifolia. Amongthesix highly contaminated samples three samples tested positive for AB1. Highest AB1 was recorded from T. cordifolia (104.19 µg/kg), followed by A. calamus (13.73 µg/kg) and M. fragrans (12.02 µg/kg). CONCLUSION: Assessment of fungal and mycotoxin contamination should be a part of the quality check while selecting HDRM for manufacture of herbal products. Safe processing and storage practices are necessary.

Extracellular Xylanolytic and Pectinolytic Hydrolase Production by Aspergillus flavus Isolates Contributes to Crop Invasion.

Several atoxigenic Aspergillus flavus isolates, including some being used as biocontrol agents, and one toxigenic isolate were surveyed for the ability to produce extracellular xylanolytic and pectinolytic hydrolases. All of the tested isolates displayed good production of endoxylanases when grown on a medium utilizing larch xylan as a sole carbon substrate. Four of the tested isolates produced reasonably high levels of esterase activity, while the atoxigenic biocontrol agent NRRL 21882 isolate esterase level was significantly lower than the others. Atoxigenic A. flavus isolates 19, 22, K49, AF36 (the latter two are biocontrol agents) and toxigenic AF13 produced copious levels of pectinolytic activity when grown on a pectin medium. The pectinolytic activity levels of the atoxigenic A. flavus 17 and NRRL 21882 isolates were significantly lower than the other tested isolates. In addition, A. flavus isolates that displayed high levels of pectinolytic activity in the plate assay produced high levels of endopolygalacturonase (pectinase) P2c, as ascertained by isoelectric focusing electrophoresis. Isolate NRRL 21882 displayed low levels of both pectinase P2c and pectin methyl esterase. A. flavus appears capable of producing these hydrolytic enzymes irrespective of aflatoxin production. This ability of atoxigenic isolates to produce xylanolytic and pectinolytic hydrolases mimics that of toxigenic isolates and, therefore, contributes to the ability of atoxigenic isolates to occupy the same niche as A. flavus toxigenic isolates.

Copper induction and differential expression of laccase in Aspergillus flavus.

Aspergillus flavus was isolated from soil and exhibited laccase activity under both constitutive and copper induced conditions. Spiking the medium with 1 mM copper sulfate resulted in an increase in the activity which reached 51.84 U/mL, a distinctive protein band was detected at 60 kDa. The extracellular enzyme was purified 81 fold using gel filtration chromatography and resulted in two different laccase fractions L1 and L2, the latter had a higher enzymatic activity which reached 79.57 U/mL and specific activity of 64.17 U/µg protein. The analysis of the spectrum of the L2 fraction showed a shoulder at 330 nm which is characteristic for T2/T3 copper centers; both copper and zinc were detected suggesting that this is an unconventional white laccase. Primers of laccase gene were designed and synthesized to recover specific gene from A. flavus . Sequence analysis indicated putative laccase (Genbank ID: JF683612) at the amino acid level suggesting a close identity to laccases from other genera containing the copper binding site. Decolorization of textile waste water under different conditions showed possible application in bioremediation within a short period of time. The effect of copper on A. flavus was concentration dependent.

The Role of Adjunctive Therapies in the Management of Invasive Sino-Orbital Infection.

OBJECTIVE: Invasive sino-orbital fungal infections are life-threatening complications of immunonosupression that are difficult to treat. Currently there are no standard treatment guidelines. The most widely accepted therapy includes parenteral anti-fungal therapy and surgical debridement of sinuses with orbital exenteration, a procedure that is not only disfiguring, but may increase morbidity. Injection of retrobulbar Liposomal Amphotericin B (L-AMB) is an alternative approach that provides local administration to infected tissues. The adjunct use of anti-fungal retrobulbar injections not been extensively reviewed in treating sino-orbital infection. We are reporting the multimodal approach of using L-AMB retrobulbar injections in combination with sinus debridement, intravenous (IV) anti-fungal therapy, and hyperbaric oxygen (HBO) for the management of sino-orbital infection. METHOD & RESULTS: Review of literature of 12 cases and retrospective evaluation of one patient with sino-orbital Aspergillus flavus infection on chemotherapy for T-cell acute lymphocytic leukemia treated with retrobulbar Amphotericin B, IV anti-fungal agents, and hyperbaric oxygen therapy. Clinical characteristics, radiographic features, management techniques, and clinical outcomes are described. CONCLUSION: Retrobulbar Amphotericin B injection may be an effective adjunct to hyperbaric oxygen and parenteral anti-fungals in the control of sino-orbital fungal infections.

Copper induction and differential expression of laccase in Aspergillus flavus

Aspergillus flavus was isolated from soil and exhibited laccase activity under both constitutive and copper induced conditions. Spiking the medium with 1 mM copper sulfate resulted in an increase in the activity which reached 51.84 U/mL, a distinctive protein band was detected at 60 kDa. The extracellular enzyme was purified 81 fold using gel filtration chromatography and resulted in two different laccase fractions L1 and L2, the latter had a higher enzymatic activity which reached 79.57 U/mL and specific activity of 64.17 U/μg protein. The analysis of the spectrum of the L2 fraction showed a shoulder at 330 nm which is characteristic for T2/T3 copper centers; both copper and zinc were detected suggesting that this is an unconventional white laccase. Primers of laccase gene were designed and synthesized to recover specific gene from A. flavus. Sequence analysis indicated putative laccase (Genbank ID: JF683612) at the amino acid level suggesting a close identity to laccases from other genera containing the copper binding site. Decolorization of textile waste water under different conditions showed possible application in bioremediation within a short period of time. The effect of copper on A. flavus was concentration dependent.

Biological Activities and Identification of Bioactive Metabolite from Endophytic Aspergillus flavus L7 Isolated from Aegle marmelos.

Aegle marmelos, a well-known Indian plant with medicinal and religious importance, has been extensively used in Indian traditional medicine. The present study aimed to isolate, identify, and evaluate the biological activities of endophytic fungi from A. marmelos. One of the isolates, labeled as L7, was identified as Aspergillus flavus using morphology and ITS gene sequence. Total phenolic and flavonoid contents in the culture filtrate were found to be 65.77 mg GAE/ml and 158.33 mg quercetin/ml of crude extract, respectively. The extract showed excellent antimicrobial activity against common human bacterial and fungal pathogens. The test extract at 700 µg/ml, which notably reduced the concentration of DPPH-free radical as percent DPPH scavenging activity, was found to be the highest (64.53 %). The extract, at the concentration of 2 mg/ml, produced 70 % inhibition of hemolysis of RBCs compared to 78 % produced by standard drug (Ibuprofen). Chemical profiling of the fermented extract using TLC followed by UV and FTIR revealed the presence of flavonoids. The HPLC analysis confirmed the presence of bioflavonoid rutin in the extract. To the best of our knowledge, this is the first report on production of bioactive flavonoid by endophytic Aspergillus flavus obtained from A. marmelos and its pharmaceutical potential. In conclusion, the endophytic Aspergillus flavus obtained from the A. marmelos could be explored as an economic and potential natural resource with diverse pharmaceutical and biological activities.

Efficiency of Artemisia nilagirica (Clarke) Pamp. essential oil as a mycotoxicant against postharvest mycobiota of table grapes.

BACKGROUND: In order to get a potent botanical fungicide for the management of fungal decay of table grapes, an experiment was conducted in which 20 essential oils of higher plants were screened at 0.33 µL mL(-1) against dominant fungi causing decay of table grapes, including Aspergillus flavus, A. niger and A. ochraceus. Furthermore, the minimum inhibitory/fungicidal concentration, fungitoxic spectrum and mycotoxin inhibition activity of the most potent oil were determined. The efficacy of the most potent oil in preservation of table grapes, along with organoleptic evaluation, was also carried out by storing 1 kg of grapes in the oil vapour. RESULTS: Artemisia nilagirica oil was found to be most toxic, exhibiting 100% mycelia inhibition of all test fungi. Moreover, 0.29 µL mL(-1) A. nilagirica oil was fungistatic and 0.58 µL mL(-1) was fungicidal for all tested species of Aspergillus. The oil exhibited a broad range of fungitoxicity against other grape berry-rotting fungi. Artemisia nilagirica oil completely suppressed the growth and mycotoxin (AFB1 and OTA) secretion of aflatoxigenic and ochratoxigenic strains of Aspergillus at 1.6 µL mL(-1) . During the in vivo experiment, fumigation of 1 kg of table grapes with 200 and 300 µL dosage of A. nilagirica oil enhanced the shelf life for up to 9 days. The oil did not show any phytotoxic effect. Besides, oil application did not substantively change the sensory properties of the fruits. CONCLUSION: Artemisia nilagirica oil can be used as an alternative botanical fungicide for the control of fruit-rotting fungi of stored grapes.

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.

Transcriptomic profiles of Aspergillus flavus CA42, a strain that produces small sclerotia, by decanal treatment and after recovery.

Aspergillus flavus is a ubiquitous saprophyte and is capable of producing many secondary metabolites including the carcinogenic aflatoxins. The A. flavus population that produces small sclerotia (S strain) has been implicated as the culprit for persistent aflatoxin contamination in field crops. We investigated how the plant volatile decanal, a C10 fatty aldehyde, affected the growth and development of the S strain A. flavus. Decanal treatment yielded fluffy variants lacking sclerotia and conidia and exhibiting a dosage-dependent radial colony growth. We used RNA-Seq analysis to examine transcriptomic changes caused by decanal and after removal of decanal. Mature sclerotia contained only 80% of the total transcripts detected in all samples in comparison to 94% for the decanal treated culture. Gene ontology (GO) analysis showed that decanal treatment increased expression of genes involved in oxidoreductase activity, cellular carbohydrate metabolism, alcohol metabolism and aflatoxin biosynthesis. The treatment affected cellular components associated with cell wall, and gene expression of glucanases, α-amylases, pectinesterase and peptidase required for its biosynthesis was increased. After decanal was removed, the culture resumed sclerotial production. Moreover, its GO terms significantly overlapped with those of the untreated culture; five of the enriched molecular functions, oxidoreductase activity, monooxygenase activity, electron carrier activity, heme binding, and iron binding were found in the untreated culture. The GO term of cellular component enriched was mainly integral protein constituents of the membrane. The results suggested that decanal halted development at the vegetative state rendering the fungus unable to produce conidia and sclerotia. The induced fluffy phenotype could be related to lower transcript abundance of flbB, flbD, and flbE but not to veA expression. Increased abundance of the laeA transcript in the treated culture correlated with early transcriptional activation of aflatoxin and kojic acid biosynthesis gene clusters. Expression profiles revealed subtle differences in timing of activation of the respective 55 secondary metabolite gene clusters.

In vitro comparative analysis of monocrotophos degrading potential of Aspergillus flavus, Fusarium pallidoroseum and Macrophomina sp.

Fungal degradation is emerging as a new powerful tool for the removal of potent neurotoxin pesticide, monocrotophos. Therefore, the present study is aimed at comparative characterization of monocrotophos degrading ability of three different fungal strains. Fungal strains were isolated from local agricultural soil by enrichment culture method, screened by gradient culture and identified as Aspergillus flavus, Fusarium pallidoroseum and Macrophomina sp. Growth kinetics revealed a direct positive influence of monocrotophos on the viability of fungal isolates. Fungal degradation was studied in phosphorus free liquid culture medium supplemented with 150 mg L(-1) concentration of monocrotophos for a period of 15 days under optimized culture conditions. Degradation of MCP followed first order kinetics with kdeg of 0.007, 0.002 and 0.005 day(-1) and half life (t1/2) of 4.21, 12.64 and 6.32 days for A. flavus, F. pallidoroseum and Macrophomina sp. respectively. To the best of our knowledge, it is the first report signifying the potential of monocrotophos degradation by Fusarium and Macrophomina sp. The results were further confirmed by HPTLC and FTIR which indicates disappearance of monocrotophos by hydrolytic cleavage of vinyl phosphate bond. Degradation of monocrotophos by fungal isolates was accompanied by the release of extracellular alkaline phosphatases, inorganic phosphates and ammonia. The overall comparative analysis followed the order of A. flavus > Macrophomina sp. > F. pallidoroseum. Therefore, it could be concluded from the study that these three different fungal strains could be effectively used as a potential candidate for the removal of monocrotophos from contaminated sites.

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 asAspergillus 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% forPenicillium 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.

Effects of amphotericin B on Aspergillus flavus clinical isolates with variable susceptibilities to the polyene in an experimental model of systemic aspergillosis.

OBJECTIVES: The aim of the present study was to evaluate the effects of amphotericin B (AMB) on clinical isolates of Aspergillus flavus. METHODS: MICs of both standard AMB and liposomal AMB (L-AMB) were determined using a broth dilution method for seven isolates of A. flavus. AMB MICs were also determined using the Etest. The activity of the polyene was then investigated in a murine model of systemic aspergillosis in which animals were infected intravenously, treated intravenously with several doses of the polyene (1-10 mg/kg/day) and observed for survival. RESULTS: Broth dilution AMB, broth dilution L-AMB and Etest AMB MICs ranged from 0.5 to 2.0 mg/L, 0.06 to >16 mg/L and 1.0 to >32 mg/L, respectively. There were two isolates for which all doses were effective at prolonging the survival. Their AMB MICs were ≤1.0 mg/L, regardless of the method/drug formulation utilized for testing. There were four isolates for which no regimen was effective. Their broth dilution AMB, broth dilution L-AMB and Etest AMB MICs ranged from 1.0 to 2.0 mg/L, 0.06 to >16 mg/L and 2.0 to >32 mg/L, respectively. There was one isolate for which only L-AMB given at 10 mg/kg/day was effective; broth dilution MICs of AMB and L-AMB were 0.5 mg/L, while the Etest MIC of AMB was 2.0 mg/L. CONCLUSIONS: Our data indicate that not all isolates of A. flavus should be considered resistant to AMB. The Etest represented the in vitro method that best correlated with the experimental infection. Finally, a clinical isolate showing an MIC ≥2.0 mg/L may be reasonably considered resistant in vivo to any dose/formulation of the polyene.

Antifungal action of Pichia anomala against aflatoxigenic Aspergillus flavus and its application as a feed supplement.

BACKGROUND: The disastrous problem of animal feed contamination by mycotoxigenic fungi continues to challenge researchers and health overseers worldwide. With the aim of preventing Aspergillus flavus growth in vitro and in corn feed, the yeast Pichia anomala was examined as a biocontrol agent. RESULTS: The yeast strain could efficiently prohibit the growth of A. flavus. P. anomala was able to produce exo-chitinase and ß-1,3-glucanase which could be suggested as a mode of action for its antifungal activity. Scanning electron microscopy of cultured P. anomala with fungal hypha revealed that A. flavus was colonised by the biocontrol yeast which subsequently led to complete hyphal lysis and deterioration. The supplementation with of P. anomala cells, as a protein source, led to an obvious increase in animals' weight gain and protein content in feed grain. Moreover, after consumption of P. anomala-supplemented feed, there was a remarkable decrease in the mortality rate among fed animals. CONCLUSION: P. anomala could be strongly recommended as a biocontrol agent against A. flavus which contaminates animal feed. Furthermore, the application of yeast cells, as a feed additive, proved its efficiency for escalating protein content and enhancing animal performance.

Equisetum arvense hydro-alcoholic extract: phenolic composition and antifungal and antimycotoxigenic effect against Aspergillus flavus and Fusarium verticillioides in stored maize.

BACKGROUND: Maize is a very important cereal for human and animal diet, but it can be contaminated by moulds and their mycotoxins. On the other hand, natural plant products with antimicrobial properties could possibly used to control mycotoxigenic fungi in foods and feeds. In this study, Equisetum arvense extract was tested for the efficacy on Aspergillus section Flavi and Fusarium section Liseola growth. Natural contaminated maize was used in this study and extract was added under different water activities (a(w)) - 0.90 and 0.95 - for Aspergillus section Flavi and Fusarium section Liseola, respectively. Moulds were inoculated in maize and incubated during 30 days. RESULTS: We confirm that E. arvense extract may be effective for the inhibition of Aspergillus section Flavi in maize with high levels of this mould. Moreover, this extract showed a good inhibition of growth on Fusarium section Liseola levels. Aflatoxin and fumonisin production was not affected by the extract. CONCLUSIONS: E. arvense extract could be an alternative to synthetic fungicides to control maize mycobiota level in moist grain.

[Screening and identification of microorganisms for decolorization of molasses spent wash].

Microorganisms were screened from the natural environment for decolorization of molasses spent wash, and the isolated strains were then employed in the treatment of actual wastewater. The primary screening was carried out on agar plates supplemented with synthesized melanoidin as the target substrate, since melanoidin is one of the most refractory pigments in wastewater. Promising microorganisms were further selected through secondary screening by decolorization of untreated actual wastewater in shaking flask cultures. Gel filtration chromatography was used to determine the molecular weight distribution of pigments in molasses spent wash before and after decolorization. A strain named A5P1 was isolated from the soil samples collected, showing a good ability of decolorizing molasses spent wash, and was later identified as Aspergillus flavus by morphology and ITS sequence analysis. Experimental study of factors affecting the decolorization performance of strain A5P1 gave the optimal conditions as follows: 4.3 x 10(4) mL(-1) of inoculum size, medium with initial pH of 4.5 and cultivation at 39 degrees C. It could decolorize 53.0% of the pigments in the untreated molasses spent wash and decreased 80% of chemical oxygen demand after four-day incubation. The result of gel filtration chromatography demonstrated that both the large and small molecular weight fractions of pigments in the molasses spent wash could be removed by strain A5P1. Based on the measurement of enzyme activities, at least three different kinds of enzymes, i. e. the enzyme with H2O2-producing activity, laccase and manganese peroxidase were involved in the decolorization process. Therefore, the decolorization mechanism of strain A5P1 was preliminarily considered to be mainly biodegradation, with bioadsorption as a minor reaction.

Efficacy of aqueous garlic extract on growth, aflatoxin B1 production, and Cyto-morphological aberrations of Aspergillus flavus, causing human ophthalmic infection: topical treatment of A. flavus keratitis

By using agar well diffusion assay, antifungal activity of aqueous extract prepared from Egyptian garlic (Allium sativum L.) was evaluated in vitro against two strains of Aspergillus flavus (OC1 and OC10) causing human ocular infection. The recorded minimum inhibitory concentration (MIC) for growth inhibition of both strains was 3.60 mg/ml. Aqueous garlic extract (AGE) was used in successive in vivo tests as an attempt to cure rabbit's fungal keratitis caused by A. flavus OC1. Findings showed that diluted preparation of AGE was effective topical antifungal agent and succeeded to cure severe A. flavus keratitis in a time course less than 10 days without any observable side effects. Microscopic examination showed that AGE induced deleterious cyto-morphological aberrations inA. flavus target cells. AGE applied to Czapek's broth via contact method was more effective on growth, spores and aflatoxin B1 production than AGE applied to the same broth at the same concentration via fumigation method.