Flavus Strains Research Articles

P022 Luliconazole—a novel potent imidazole activity against Aspergillus niger and Aspergillus flavus causing otomycosis

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PM Background and ObjectivesSuppurative otitis media (SOM) is characterized by the inflammation of the middle ear and mastoid, tympanic membrane perforation as well as discharge. The tympanic membrane perforation may result in increased exposure of the middle ear to pathogens. Aspergillus niger and Aspergillus flavus, are the most common causative agents of otomycosis with worldwide distribution, when it spreads from the external auditory canal to adjacent anatomical structures, it is classified as Aspergillus invasive otitis externa. Aspergillus otomycosis treatment is initiated by thorough cleaning of the ear canal, accomplished with suction, and drying with cotton swabs. In developing countries, SOM is a major cause of preventable hearing loss, its incidence ranges from 7% to 46% and is common amongst children of lower socioeconomic status. Treatment of SOM is directed at debridement and drying the ear with topical antifungal agents. Extensive surgical debridement and systemic antifungal therapy are needed in cases of refractory otomycosis or Aspergillus invasive otitis externa. Despite this management, treatment failure may result from suboptimal therapeutic management caused by antifungal agent toxicity. Luliconazole is currently confirmed for the topical therapy of dermatophytosis. Moreover, it is found that luliconazole has in vitro activity against some molds and yeast species. The aim of the present study was to evaluate the efficacy of luliconazole in comparison to routinely used antifungals on clinical isolates of A. niger and A. flavus.MethodsThe study was carried out in the Department of Microbiology, SRIHER, Chennai. A total of 55 (29 A. niger and 26 A. flavus) strains of Aspergillus isolates obtained from clinical otomycosis cases were confirmed based on macroscopic and microscopic identification by Lacto Phenol Cotton Blue mount and slide culture technique. Antifungal susceptibility patterns of all the Aspergillus isolates to itraconazole, voriconazole, posaconazole, and luliconazole were determined by broth microdilution method as per Clinical Laboratory Standards Institute (CLSI) M38-A2 guidelines.ResultsThe lowest minimum inhibitory concentration (MIC) geometric mean (GM) (0.00309 μg/ml) was attributed to luliconazole followed by posaconazole (0.18409 μg/ml), voriconazole (1.02727 μg/ml) and itraconazole (11.0091 μg/ml). Also, among the azoles tested, luliconazole had the lowest MIC50 and MIC90 values of 0.00098 μg/ml and 0.00781 μg/ml respectively. Among the triazoles tested posaconazole had a lower MIC50 and MIC90 values of 0.125 μg/ml and 0.25 μg/ml. Being the drug of choice for invasive aspergillosis voriconazole had a slightly higher MIC50 and MIC90 value of 1 μg/ml and 2 μg/ml. Luliconazole was found to be more effective even for pan azole-resistant isolates (n = 3) with lower MIC values.ConclusionThe results of this study showed that luliconazole had an excellent in vitro activity against all Aspergillus isolates with a lower MIC GM, MIC50, and MIC90 values than the triazoles tested. Hence, this novel imidazole antifungal agent can be regarded as appropriate Candidate for the treatment of otomycosis caused by A. niger and A. flavus strains. Also, luliconazole showed better efficacy with lower MIC values for pan azole resistant isolates, suggesting that it could be a potential antifungal for treating aspergillosis caused by pan azole-resistant isolates.

Comparative Analysis of Compost Quality Produced from Fungal Consortia and Rice Straw by Varying C/N Ratio and its Effect on Germination of Vigna radiata

Composting is considered to be one of the best methods for handling organic waste. It is a natural process and takes months to give quality mature compost. Characteristics of initial wastes and process conditions are the compost maturity deciding factors. Some biological inoculants, e.g., bacteria and fungi, can reduce the compost time and improve its quality. This research is based on the hypothesis that using fungus consortia on rice straw will boost the activities of microbes and, as a result, the rate of composting. The hypothesis was tested by preparing compost using rice straw residue with and without the applications of fungal consortia. Fungal consortia of Aspergillus flavus, Aspergillus fumigatus, and Aspergillus terreus cellulose-degrading strains along with Pusa-1121 rice variety were used for the study. Different C/N ratios were achieved by varying rice straw, green leaves, poultry droppings, and fungal inoculant proportions. During various stages of composting, changes in total nitrogen, organic carbon, C/N ratios, and other parameters were calculated. The germination index of Mung beans (Vigna radiata) was used to measure the quality of the completed compost extract. Statistical analysis with the help of a two-tailed independent t-test at the confidence level of 95% was applied to determine the statistical difference between the treatments and control. It has been found that the Seed Germination index of treatment C/N 30 was 91.5% and that of C/N 26 was 79.1% which were significantly (p<0.01) different from the 54% GI of control.

Pre-harvest strategy for reducing aflatoxin accumulation during storage of maize in Argentina

Maize (Zea mays L.) is an important crop in Argentina. Aspergillus flavus may infect this crop at growing stage and the harvested kernels can be contaminated with aflatoxins (AFs), whose levels may increase during storage. In Argentina, silo bags, a hermetic type of storage system, are widely used. Biocontrol based on competitive exclusion by atoxigenic A. flavus strains is a useful tool for AFs management at pre-harvest stage. The aim of the present study was to evaluate the effect of pre-harvest biocontrol treatments on aflatoxin B1 (AFB1) accumulation in maize stored in silo bags during 3 and 6 months. Three bioformulations based on A. flavus AFCHG2 and ARG5/30 strains were applied during field trials as single and mixed inocula. Harvested kernels were stored in non-hermetic and hermetic silo bags. At initial time (t0), 3 and 6 months (t3 and t6) the following parameters were evaluated: percentage of damaged kernels, moisture content, water activity, Aspergillus section Flavi incidence, relative humidity, O2 and CO2 levels into the silo bags, and AFB1 levels. The biocontrol strains included in the 3 bioformulations were able to infect maize kernels during the field trial and displaced native toxigenic isolates. At t0 control plots showed 10.9 ± 0.4 μg/kg of AFB1 while no AFs were detected in all the treatments. Along the storage assay AFB1 levels varied from not detected (<1 μg/kg) to 20.1 ± 0.8 μg/kg. Hermetic bags were better than non-hermetic bags in preventing AFB1 accumulation. Both single and mixed inocula were effective to control AFB1 accumulation in maize kernels during 3 and 6 months. AFB1 was not detected in kernels from the treatment at field stage with AFCHG2 + ARG5/30 after 6 months of storage into hermetic bags. The application of the biocontrol agents at field stage is an appropriate tool to reduce AFB1 accumulation under storage in hermetic silo bags. This is the first report on biocontrol strategy based on native atoxigenic strains applied at pre-harvest stage to reduce AFB1 accumulation during storage in Argentina.

Set2 family regulates mycotoxin metabolism and virulence via H3K36 methylation in pathogenic fungus Aspergillus flavus

ABSTRACT Aspergillus flavus infects various crops with aflatoxins, and leads to aspergillosis opportunistically. Though H3K36 methylation plays an important role in fungal toxin metabolism and virulence, no data about the biological function of H3K36 methylation in A. flavus virulence has been reported. Our study showed that the Set2 histone methyltransferase family, AshA and SetB, involves in morphogenesis and mycotoxin anabolism by regulating related transcriptional factors, and they are important for fungal virulence to crops and animals. Western-blotting and double deletion analysis revealed that AshA mainly regulates H3K36me2, whereas SetB is mainly responsible for H3K36me3 in the nucleus. By construction of domain deletion A. flavus strain and point mutation strains by homologous recombination, the study revealed that SET domain is indispensable in mycotoxin anabolism and virulence of A. flavus, and N455 and V457 in it are the key amino acid residues. ChIP analysis inferred that the methyltransferase family controls fungal reproduction and regulates the production of AFB1 by directly regulating the production of the transcriptional factor genes, including wetA, steA, aflR and amylase, through H3K36 trimethylation in their chromatin fragments, based on which this study proposed that, by H3K36 trimethylation, this methyltransferase family controls AFB1 anabolism through transcriptional level and substrate utilization level. This study illuminates the epigenetic mechanism of the Set2 family in regulating fungal virulence and mycotoxin production, and provides new targets for controlling the virulence of the fungus A. flavus. AUTHOR SUMMARY The methylation of H3K36 plays an important role in the fungal secondary metabolism and virulence, but no data about the regulatory mechanism of H3K36 methylation in the virulence of A. flavus have been reported. Our study revealed that, in the histone methyltransferase Set2 family, AshA mainly catalyzes H3K36me2, and involves in the methylation of H3K36me1, and SetB mainly catalyzes H3K36me3 and H3K36me1. Through domain deletion and point mutation analysis, this study also revealed that the SET domain was critical for the normal biological function of the Set2 family and that N455 and V457 in the domain were critical for AshA. By ChIP-seq and ChIP-qPCR analysis, H3K36 was found to be trimethylation modified in the promotors and ORF positions of wetA, steA, aflR and the amylase gene (AFLA_084340), and further qRT-PCR results showed that these methylation modifications regulate the expression levels of these genes. According to the results of ChIP-seq analysis, we proposed that, by H3K36 trimethylation, this methyltransferase family controls the metabolism of mycotoxin through transcriptional level and substrate utilization level. All the results from this study showed that Set2 family is essential for fungal secondary metabolism and virulence, which lays a theoretical groundwork in the early prevention and treatment of A. flavus pollution, and also provides an effective strategy to fight against other pathogenic fungi.

MORPHOCULTURAL CHARACTERIZATION AND QUALITATIVE ASSESSMENT OF AFLATOXINS PRODUCING Aspergillus flavus ASSOCIATED WITH Arachis hypogaea IN PUNJAB, PAKSITAN

Aflatoxins, produced by ubiquitous Aspergillus section Flavi species, are polyketide pathway-derived secondary metabolites and, due to their highly carcinogenic, mutagenic, and teratogenic nature, pose human severe health issues globally. In this study, raw groundnut kernels were collected from groundnut growing districts of Punjab, Pakistan, at pre and post-harvest stages. Groundnut-associated Aspergillus flavus isolates were morphologically identified, and their respective aflatoxins producing potential was determined through cultural methods; UV illumination and ammonia vapor exposure. Three hundred isolates were cultured on differential media, Malt extract agar, and Sabouraud dextrose agar, for studying typical morphocultural characteristics, and detailed microscopy. Observed phenotypic variability assorted isolates into twenty groups. Representative isolate of each group was grown on coconut agar medium and exposed to UV illumination and ammonia vapors to observe possible fluorescence excitation and reverse colony color change, respectively. Results of both cultural methods conformed to each other; 35% of isolates produced fluorescent ring upon UV exposure and pinkish reverse colony color upon ammonia vapors exposure (toxigenic), 50% isolates showed no change (atoxigenic), and 15 % showed unclear results. This study provides evidence that groundnut kernels are highly exposed to toxigenic A. flavus strains both at pre and post-harvest stages.

Influence of abiotic factors (water activity and temperature) on growth and aflatoxin production by Aspergillus flavus in a chickpea-based medium

The effect of water activity (aW; 0.87, 0.90, 0.92, 0.94, 0.96, 0.98 and 0.99), temperature (15, 25, and 30 °C), incubation time (5, 10, 14, and 21 days), and their interactions on mycelial growth and aflatoxin production in a chickpea-based medium by three Aspergillus flavus strains isolated from chickpea grains in Argentina was evaluated. Maximum growth rates were obtained at the highest aW (0.99) and 30 °C, with growth decreasing as the aW of the medium was reduced. Maximum levels of aflatoxins were produced at 0.99 aW and 25 °C after 5 days of incubation for two strains, and at 25 °C and 0.96 aW after 21 days of incubation for the third strain. The aflatoxin concentrations varied considerably depending on the aW and temperature interactions assayed. Two-dimensional profiles of aW by temperature interactions were developed from these data to identify areas where conditions indicate a significant risk from aflatoxin accumulation on chickpea. This study provides useful baseline data on conditions representing a high and a low risk for contamination of chickpea by aflatoxins which is of greater concern because this pulse is destined mainly for human consumption.

Assessment of the Potential of a Native Non-Aflatoxigenic Aspergillus flavus Isolate to Reduce Aflatoxin Contamination in Dairy Feed.

Aspergillus species can produce aflatoxins (AFs), which can severely affect human and animal health. The objective was to evaluate the efficacy of reducing AF contamination of a non-aflatoxigenic isolate of A. flavus experimentally coinoculated with different aflatoxigenic strains in whole plant (WP), corn silage (CS), immature grains (IG) and in culture media (CM). An L-morphotype of A. flavus (CS1) was obtained from CS in a dairy farm located in the Mexican Highland Plateau; The CS1 failed to amplify the AFs biosynthetic pathway regulatory gene (aflR). Monosporic CS1 isolates were coinoculated in WP, CS, IG and CM, together with A. flavus strains with known aflatoxigenic capacity (originating from Cuautitlán and Tamaulipas, Mexico), and native isolates from concentrate feed (CF1, CF2 and CF3) and CS (CS2, CS3). AF production was evaluated by HPLC and fungal growth rate was measured on culture media. The positive control strains and those isolated from CF produced a large average amount of AFs (15,622 ± 3952 and 12,189 ± 3311 µg/kg), whereas A. flavus strains obtained from CS produced a lower AF concentration (126 ± 25.9 µg/kg). CS1 was efficient (p < 0.01) in decreasing AF concentrations when coinoculated together with CF, CS and aflatoxigenic positive control strains (71.6–88.7, 51.0–51.1 and 63.1–71.5%) on WP, CS, IG and CM substrates (73.9–78.2, 65.1–73.7, 63.8–68.4 and 57.4–67.6%). The results suggest that the non-aflatoxigenic isolate can be an effective tool to reduce AF contamination in feed and to minimize the presence of its metabolites in raw milk and dairy products intended for human nutrition.

Medium for the Production of Bacillus-Based Biocontrol Agent Effective against Aflatoxigenic Aspergillus flavus: Dual Approach for Modelling and Optimization.

One of the leading limiting factors for wider industrial production and commercialization of microbial biopesticides refers to the high costs of cultivation media. The selection of alternative sources of macronutrients crucial for the growth and metabolic activity of the producing microorganism is a necessary phase of the bioprocess development. Gaining a better understanding of the influence of the medium composition on the biotechnological production of biocontrol agents is enabled through bioprocess modelling and optimization. In the present study, after the selection of optimal carbon and nitrogen sources, two modelling approaches were applied to mathematically describe the behavior of the examined bioprocess—the production of biocontrol agents effective against aflatoxigenic Aspergillus flavus strains. The modelling was performed using four independent variables: cellulose, urea, ammonium sulfate and dipotassium phosphate, and the selected response was the inhibition-zone diameter. After the comparison of the results generated by the Response Surface Methodology (RSM) and the Artificial Neural Network (ANN) approach, the first model was chosen for the further optimization step due to the better fit of the experimental results. As the final investigation step, the optimal cultivation medium composition was defined (g/L): cellulose 5.0, ammonium sulfate 3.77, dipotassium phosphate 0.3, magnesium sulfate heptahydrate 0.3.

Light and scanning electron microscopic characterization of aflatoxins producing Aspergillus flavus in the maize crop.

Maize (Zea mays L.) is considered as one of the main cereals, used as a source of food, forage, and processed products. The loss of maize productivity is reported due to effect on roots, stalks, ears, and kernels mainly caused by many fungi. Among these fungal pathogens of maize, Aspergillus flavus (A. flavus) are the most prevalent that produces highly toxigenic aflatoxins that are highly carcinogenic to the consumers. The present study is confined to isolate and characterize the A. flavus from maize seeds for accurate identification that can be helpful for determination and management of aflatoxins in maize crop. Eighty stored seed samples of maize were collected from warehouses where seeds are stored for food and feeding purposes. For the isolation of A. flavus, Potato Dextrose Agar was used. Isolated fungi were identified macro and microscopically using light microscope and scanning electron microscope. A total of 212 Aspergillus isolates were identified based on macro-morphological and micro-morphological characteristics. The results showed that A. flavus colonies were granular, flat with yellow-green to deep yellow-green colony color having a white border and compact, spherical spore heads. Rapid rate of growth was observed maturing in about 3-5 days. In microscopic features, A. flavus have apically swollen conidiophores with various conidia bearing cells in long and dry chains. Spherical conidial heads were split into several columns ranging 300-400 μm in diameter. This will be helpful for farmers, researchers and traders in future for correct identification of sources of aflatoxins. RESEARCH HIGHLIGHTS: Maize seed samples were collected from Pothohar region of Pakistan. The fungi were isolated on PDA. Aspergillus flavus was identified macro-morphologically by observing growth rate, colony color and texture. Furthermore, these fungi were identified micro-morphologically by using light and scanning electron microscope. The 212 Aspergillus flavus strains were isolated and identified.

Effect of Voriconazole on Biofilm of Filamentous Species Isolated from Keratitis

Background: Biofilm formation is a vital feature of the pathogenesis in filamentous fungi. Objectives: Herein, we investigated in vitro antifungal pattern of biofilm of filamentous species keratitis isolates, and the effect of different concentrations of voriconazole on biofilm morphology changes using scanning electron microscopy. Methods: Here 25 ocular fungal isolates were included (Fusarium solani; (n = 15) and Aspergillus flavus; (n = 10). We determined the biofilm formation of isolates in terms of their susceptibilities to different antifungals on sessile cells. This procedure was performed according to CLSI-M38-3rd. Biofilm morphology changes revealed with scanning electron microscopy (SEM). Results: Twelve F. solani strains displayed the capacity to form biofilms from patients wearing contact lenses, while 8 A. flavus strains were recovered from cornea scrapings of trauma. Itraconazole, posaconazole and natamycin had the maximum activity against biofilms of all tested filamentous strains (MIC ranging from 0.031 - 0.25 µg/mL, 0.031 - 0.25 µg/mL and 2 - 4 µg/mL), respectively. Three F. solani and one A. flavus strains showed the high MIC values against voriconazole (MIC ≥ 1 µg/mL). Posaconazole revealed the lowest MIC values against biofilms of two strains (MIC ranging from 0.031 - 0.25 µg/mL); however, no significant difference was observed for itraconazole (P &gt; 0.05). The efficacy of 16 µg/mL and 4 µg/mL of voriconazole was confirmed for biofilm of F. solani and A. flavus, respectively. The considerable changes in the morphologies of improved biofilms were seen by SEM. Conclusions: For the successful treatment of fungal biofilm infections, it was necessary to have knowledge of the mechanism of action, penetration rates, and therapeutic concentrations of drugs.

Efficacy of sodium metabisulphite for control of Aspergillus flavus and aflatoxin B1 contamination in vitro and in chilli powder and whole red chillies

Chillies are an important spice in many regions of the world. During the production and processing phases, they are prone to infection by mycotoxigenic fungi, especially Aspergillus Section Flavi species and contamination with aflatoxins. There is significant interest in controlling aflatoxin B1 (AFB1) contamination in such spices to ensure they remain below the legislative limits for human consumption. This study initially examined the potential efficacy of sodium metabisulphite (NaMBS, up to 5000 mg/L) for the control of growth and AFB1 production by Aspergillus flavus strains in vitro on a chilli-based medium modified to 0.93–0.995 water activity, aw). Subsequent studies examined efficacy of NaMBS in stored naturally contaminated chilli powder and whole red chillies, or when inoculated with A. flavus at 0.70, 0.80, 0.90, 0.95 aw and 30 °C for 20 days. NaMBS was effective in vitro in controlling growth of the three strains of A. flavus at the different aw levels examined. No growth occurred with >500–1250 NaMBS mg/L. AFB1 was only produced at 0.98 aw with complete inhibition at ≥1250 mg/L of the preservative at 30 °C. The in vitro ED50, ED90 for NaMBS varied with aw conditions. In situ studies with chilli powder and whole red dry chillies (naturally contaminated or + conidial inoculum of A. flavus) showed that at 0.70 and 0.80 aw, NaMBS treatments of 1000 and 2000 mg/L resulted in AFB1 contamination levels below the legislative limits for spices. However, under wetter conditions at 0.90 and 0.95 aw, AFB1 contamination of stored chilli powder, −/+ A. flavus inoculum, were above the legislative limits, even with 2000 mg/L NaMBS and 20 days storage at 30 °C. Stored whole chillies inoculated with A. flavus resulted in a reduction of populations, especially at 2000 mg/L NaMBS. Control of AFB1 was achieved only with 2000 mg/L NaMBS treatment after 10 and 20 days storage. Studies with commercial laminated sheets containing immobilised NaMBS with slow release of SO2 properties significantly reduced fungal populations and effectively controlled AFB1 contamination of the stored and packaged whole red chillies.

The Analysis and Experimentation of Aspergillus flavus Colonizing Corn in West Tennessee

Aspergillus flavus is an opportunistic fungus that affects many crops including corn. A. flavus colonizes the ear of the corn, causing a disease known as Aspergillus ear rot (AER). AER contributes to yield loss in two ways; through fungal infection, which results in decreased kernel weight, and through contamination of the grain with aflatoxins. A. flavus has the ability to produce toxic secondary metabolites called aflatoxins which are harmful to humans and animals when consumed. Contamination with aflatoxin can result in reduced price, or rejection of grain depending on FDA regulatory limits. Due to their ability to compete with and displace toxigenic strains, atoxigenic (non‐toxin producing) strains of A. flavus have been used as a biological control, such as the Syngenta product Afla‐Guard®. The evaluation of local populations of A. flavus strains enables researchers to gather information about what is present in the field (atoxigenic or toxigenic) and can help make more informed management recommendations. Therefore, the purpose of this research was to evaluate the characteristics of an unknown isolate of A. flavus isolated from corn in West Tennessee in comparison to known atoxigenic (1 isolate from Afla‐Guard®) and toxigenic isolates (2 isolates). Each isolate was grown on barley seed and plated onto MDRB media three times (i.e. 3 replications) along with a negative control (autoclaved barley) (total of 15 plates). Radial growth and colony morphology of each isolate were evaluated at 3‐ and 5‐days after plating in a laboratory setting. Based on observations of the activity of the TN field collected isolate, it was predicted to grow slower than the Afla‐Guard®/atoxigenic isolate. Preliminary results demonstrate that the Afla‐Guard®/atoxigenic isolate did outgrow the TN strain as hypothesized. Additional research is required to determine the toxicity of the TN strain, as well as evaluation of other naturally occurring strains in TN in relation to potential use as biological controls.

Survival and growth of microscopic fungi derived from tropical regions under future heat waves in the Pannonian Biogeographical Region

Warming and heat waves are predicted by different climate models in the near future in the Pannonian Biogeographical Region (PBR). These climatic effects may have impact on the prevalence and distribution of certain fungal species of this area. In this study the effects of predicted climate scenarios were tested on fungi being endemic or unintentionally introduced by global trade from regions of warm temperate climate. Common fungal species were selected for the study and exposed to heat waves during 7 days according to two climate scenarios: one moderately (RCP 4.5, Tavg = 27 °C, Tmax = 35 °C, RH: 100%) and one strongly pessimistic (RCP 8.5, Tavg = 30 °C, Tmax = 40 °C, RH: 100%) that include predictions for the Central Hungarian Region for July 2050. According to our results, Aspergillus flavus, Aspergillus niger, Aspergillus tubingensis and Fusarium strains introduced from tropical regions tolerated heat waves, unlike Penicillium and Talaromyces spp. and endemic Cladosporium spp. which were unable to grow under the RCP 8.5 treatment. The effects of climate change on fungi raise new issues not only from economic and health perspectives, but also in relation with plant protection and environment. Our results suggest that heat waves driven by climate change promote the colonization and growth of the tested strains of non-native fungi more likely than that of the native ones.

Fungal Contamination and Characterization of Aspergillus flavus on Poultry Feeds and their Ingredients in North Sumatera

Poultry feeds mainly consist of cereal grains and crops that are vulnerable to fungal infection. Improper handling of the feeds increases spoilage and contamination by aflatoxigenic fungi. The aim of this study was to determine fungal contamination including characterization of toxigenic and non-toxigenic Aspergillus flavus strains in poultry feed and its ingredients. We collected 200 composite samples of compound poultry feeds and their ingredients sold by retailers at traditional markets. The fungal population was enumerated using a dilution followed by the pour plate in dichloran 18% glycerol agar medium. All isolated A. flavus were identified by culture technique and molecular approach. Aflatoxin production was determined in 10% coconut agar medium with thin layer chromatography. Regulatory (aflR) and structural (nor-1, ver-1, omt-1) genes of A. flavus strains were isolated using four sets of primers (aflR, nor-1, ver-1, omt-1). We found that all feeds were infected by fungi. Aspergillus chevalieri was the most found in all feeds, particularl on chick starter. Of the 200 samples of compound and poultry feed ingredients, 57 isolates of A. flavus were examined and 16 of them (28%) were toxigenic, each strain producing aflatoxin B1 from &lt;3.01 to 35.50 ppb. Among feeds, layer finisher, pellet starter and grain corn were the most infected by toxigenic A. flavus, respectively. The contamination occurred on feeds that contained corn as the main compound. Routine analysis of compound poultry feeds and their ingredients for aflatoxigenic fungi particularly A. flavus is compulsory

Characterization of Ugandan Endemic Aspergillus Species and Identification of Non-Aflatoxigenic Isolates for Potential Biocontrol of Aflatoxins.

Acute stunting in children, liver cancer, and death often occur due to human exposure to aflatoxins in food. The severity of aflatoxin contamination depends on the type of Aspergillus fungus infecting the crops. In this study, Aspergillus species were isolated from households’ staple foods and were characterized for different aflatoxin chemotypes. The non-aflatoxigenic chemotypes were evaluated for their ability to reduce aflatoxin levels produced by aflatoxigenic A. flavus strains on maize grains. Aspergillus flavus (63%), A. tamarii (14%), and A. niger (23%) were the main species present. The A. flavus species included isolates that predominantly produced aflatoxins B1 and B2, with most isolates producing a high amount (>20 ug/µL) of aflatoxin B1 (AFB1), and a marginal proportion of them also producing G aflatoxins with a higher level of aflatoxin G1 (AFG1) than AFB1. Some non-aflatoxigenic A. tamarii demonstrated a strong ability to reduce the level of AFB1 by more than 95% when co-inoculated with aflatoxigenic A. flavus. Therefore, field evaluation of both non-aflatoxigenic A. flavus and A. tamarii would be an important step toward developing biocontrol agents for mitigating field contamination of crops with aflatoxins in Uganda.

Molecular inclusion of Cymbopogon martinii essential oil with β-cyclodextrin as a strategy to stabilize and increase its bioactivity

An essential oil complex (EOP) of Cymbopogon martinii with β-cyclodextrin (β-CD) was evaluated as a strategy to stabilize the essential oil of Palmarosa and increase its bioactivity. The complexation methods used were co-precipitation and kneading, and, for comparison, physical mixing was carried out. To confirm the molecular inclusion, techniques were combined for the characterization of the inclusion complex, such as Fourier Transformer Raman Spectroscopy (FT-Raman), Attenuated Total Reflection Fourier Transform Infrared Spectroscopy (ATR-FTIR), Differential Scanning Calorimetry (DSC) and Thermogravimetric Analysis (TGA), which suggested the formation of the EOP-β-CD complex with complexation efficiency through the co-precipitation and kneading methods of 77.68% and 47.74%, respectively. Stability of the free and complexed EOP in relation to major compounds (linalool, geraniol, geranyl acetate and caryophyllene) during 14 days of storage was analyzed through Headspace Gas Chromatography coupled with Mass Spectrometry. Geraniol showed stability over the period of evaluation and increased availability when complexed by the Co-precipitation and kneading complex. Furthermore, the antifungal and antitumor activity of the EOP-β-CD complex was evaluated, showing reduced mycelial growth against Aspergillus flavus and Fusarium verticillioides strains compared to the free EOP (percentage of inhibition to toxigenic fungi A. flavus EOP-KM: 29.41%; EOP-CP and EOP-PM: 24.37 and 14.28%, respectively, and, the free EOP with 11.76%). In addition, an interesting activity against tumor cells (HeLa and HT-29) was observed against the tested complexes, which emphasizes the importance of the biotechnological use of β-CD for the stability of natural compounds.

Quantum computational, spectroscopic investigation, molecular docking, and in vitro pharmacological studies of sulfonamide Schiff base

A new Schiff base, (E)-4-((3,5-dibromo-2-hydroxybenzylidene)amino)-N-(5-methylisoxazol-3-yl)benzenesulfonamide was synthesized and characterized by various physico-chemical, X-ray crystallography and DFT methods. The experimental findings were compared with the computed data. The characteristic azomethine band was found at 1616 cm−1 in the experimental FTIR spectrum. The double bond distance between C7=N1 was 1.277(5) Å. Single X-ray crystallography data indicated that the crystal system of the compound was monoclinic with space group P21/c. The experimental values were well correlated with the computed ones. The kinetic stability of the compound was high due to the larger HOMO-LUMO energy gap. Molecular docking and POM (Petra/Osiris/Molinspiration) investigation of the compound were also performed. POM analysis identified one antibacterial and two antitumor pharmacophore sites in the compound. It exhibited higher binding energy than the reference drug in molecular docking study. The compound followed Lipinski's rule of five and exhibited promising drug-like character and drug score. Moreover, in vitro anticancer, antibacterial, antifungal, anti-inflammatory and antioxidant properties of the compound were also carried out. It showed good antibacterial effects against S. aureus and S. typhi strains. In case of antifungal activity, the compound showed moderate inhibitory activity against both A. niger and A. flavus strains. Besides, it has the capability to stabilize the human red blood cell membrane in hypotonic solution and protect hemolysis. The highest cytotoxic activity of the compound with IC50 value of 65.344 μg/mL was observed against A549 lung cancer cells. Finally, the compound showed moderate antioxidant activity with IC50 value of 4.25 μM.

Secondary metabolites produced by solid-state fermentation of a neotropical Aspergillus flavus strain confer anti-mosquito activity with long shelf-life

Fungal metabolites have shown promising potential as alternative tools for controlling mosquito larvae. Here, we evaluated the insecticidal activities of secondary metabolites (smAf) obtained by solid-state fermentation (SSF) of maize substrate inoculated with spores of the fungi Aspergillus flavus and evaluated the smAf toxicity against Aedes aegypti larvae. We further applied computational approaches to predict the interactions of three potential constituents of smAf with the A. aegypti acetylcholinesterases, which consists of a relevant physiological target for insecticides. Our results revealed that esters [Hexadecenoic acid, methyl ester; (E, E) − 9,12-octadecadienoic acid, methyl ester; (E, E) - 11,14-eicosadecanoic acid, methyl ester; (Z, Z) − 9,12-octadecadienoic acid, ethyl ester]; chromene [2,5,5,8a-tetramethyloctahydro-2Hchromene]; fatty acids [Hexadecenoic acid] and aromatic compounds [2-hydroxy-5-methoxybenzaldehyde, propyl ether; 3-hydroxy-4-propionylphenyl acetate; N-(2,4-dimethoxyphenyl) acetamide] were present in smAf. Toxicological bioassays demonstrated that smAf exhibited similar mosquitocidal activities against A. aegypti mosquito larvae just after obtention and after 48 storage months. Our computational predictions revealed the 2,5,5,8a-tetramethyloctahydro-2 H-chromene and acetylcholinesterase as the interaction with the highest binding energy (−7.3 kcal/mol). Our findings reinforce the mosquitocidal potential of smAf and demonstrate its efficacy, even after a long storage period.

Peer review of the pesticide risk assessment of the active substance Aspergillus flavus strain MUCL54911.

The conclusions of the European Food Safety Authority (EFSA) following the peer review of the initial risk assessments carried out by the competent authority of the rapporteur Member State, Italy, for the pesticide active substance Aspergillus flavus strain MUCL54911 and the considerations as regards the inclusion of the substance in Annex IV of Regulation (EC) No 396/2005 are reported. The context of the peer review was that required by Regulation (EC) No 1107/2009 of the European Parliament and of the Council. The conclusions were reached on the basis of the evaluation of the representative use of Aspergillus flavus strain MUCL54911 as a biocontrol agent for reduction of aflatoxins contamination on maize (field application). The reliable endpoints, appropriate for use in regulatory risk assessment, are presented. Missing information identified as being required by the regulatory framework is listed. Concerns are identified.

Synthesis and biological evaluation of novel 2‐arylquinoline‐3‐fused thiazolo[2,3‐c]1,2,4‐triazole heterocycles as potential antiproliferative and antimicrobial agents

AbstractA series of novel 2‐arylquinoline‐3‐fused thiazolo[2,3‐c]1,2,4‐triazole heterocycles 9a‐t were efficiently synthesized using simple conventional methods in good yields. The structure of newly synthesized molecules was characterized on the basis of their IR, 1H NMR, 13C NMR and mass spectral data. Among 9a‐t, compounds 9h, 9n, 9b and 9d exhibited highly significant antiproliferative activity against two cancer cell lines C6 (nerve cells) and MCF‐7 (human breast adenocarcinoma cells) when compared with standard reference Doxorubicin. In vitro antimicrobial activities of target compounds 9h, 9b, 9d, 9m and 9n were effectuated on Gram‐positive Staphylococcus aurus (ATCC 25923), Bacillus subtilis (ATCC 6633) and Gram‐negative strains Klebsiella Pneumonia (ATCC 31488) and Escherichia coli (ATCC 25966) strains and found to exhibit promising activity against standard Ciprofloxacin drug. Further, when in vitro antifungal activity was conducted on Aspergillus flavus and Aspergillus niger strains 9h, 9b, 9d and 9n were exhibited potent activity when compared with standard Fluconazole drug moiety.