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Near infrared hyperspectral imaging over the spectral range of 900–2500 nm was investigated for its potential to identify maize kernels inoculated with aflatoxigenic fungus (AF13) from healthy kernels and kernels inoculated with non-aflatoxigenic fungus (AF36). A total of 900 kernels were used with 3 treatments, namely, each 300 kernels inoculated with AF13, AF36 and sterile distilled water as control, separately. One hundred kernels from each treatment of 300 kernels were incubated for 3, 5 and 8 days, to obtain diverse samples. Based on the full mean spectra extracted from the same kernel side(s), the best mean overall prediction accuracies achieved were 96.3% for the 3-class (control, non-aflatoxigenic and aflatoxigenic) classification and 97.8% for the 2-class (aflatoxigenic-negative and -positive) classification using the partial least-squares discriminant analysis (PLS-DA) method. The 3-class and 2-class models using the full mean spectra extracted from different kernel sides had the best mean overall prediction accuracies of 91.5% and 95.1%. Using the most important 30, 55 and 100 variables determined by the random frog (RF) algorithm, the simplified type I-RF-PLSDA models achieved the mean overall prediction accuracies of 87.7%, 93.8% and 95.2% for the 2-class discrimination using different kernel sides’ information. Among the most important 55 and 100 variables, a total of 25 and 67 variables were consistently selected in the 100 random runs and were therefore used further for establishing the type II-RF-PLSDA models. Using these 25 and 67 variables, the type II-RF-PLSDA models obtained the mean overall prediction accuracies of 82.3% and 94.9% separately.

Aspergillus flavus contaminates agricultural products worldwide with carcinogenic aflatoxins that pose a serious health risk to humans and animals. The fungus survives adverse environmental conditions through production of sclerotia. When fertilized by a compatible conidium of an opposite mating type, a sclerotium transforms into a stroma within which ascocarps, asci, and ascospores are formed. However, the transition from a sclerotium to a stroma during sexual reproduction in A. flavus is not well understood. Early events during the interaction between sexually compatible strains of A. flavus were visualized using conidia of a green fluorescent protein (GFP)-labeled MAT1-1 strain and sclerotia of an mCherry-labeled MAT1-2 strain. Both conidia and sclerotia of transformed strains germinated to produce hyphae within 24 h of incubation. Hyphal growth of these two strains produced what appeared to be a network of interlocking hyphal strands that were observed at the base of the mCherry-labeled sclerotia (i.e., region in contact with agar surface) after 72 h of incubation. At 5 wk following incubation, intracellular green-fluorescent hyphal strands were observed within the stromatal matrix of the mCherry-labeled strain. Scanning electron microscopy of stromata from a high- and low-fertility cross and unmated sclerotia was used to visualize the formation and development of sexual structures within the stromatal and sclerotial matrices, starting at the time of crossing and thereafter every 2 wk until 8 wk of incubation. Morphological differences between sclerotia and stromata became apparent at 4 wk of incubation. Internal hyphae and croziers were detected inside multiple ascocarps that developed within the stromatal matrix of the high-fertility cross but were not detected in the matrix of the low-fertility cross or the unmated sclerotia. At 6 to 8 wk of incubation, hyphal tips produced numerous asci, each containing one to eight ascospores that emerged out of an ascus following the breakdown of the ascus wall. These observations broaden our knowledge of early events during sexual reproduction and suggest that hyphae from the conidium-producing strain may be involved in the early stages of sexual reproduction in A. flavus. When combined with omics data, these findings could be useful in further exploration of the molecular and biochemical mechanisms underlying sexual reproduction in A. flavus.

Efforts in genome sequencing in the Aspergillus genus have led to the development of quality reference genomes for several important species including A. nidulans, A. fumigatus, and A. oryzae. However, less progress has been made for A. flavus. As part of the effort of the USDA-ARS Annual Aflatoxin Workshop Fungal Genome Project, the isolate NRRL3357 was sequenced and resulted in a scaffold-level genome released in 2005. Our goal has been biologically driven, focusing on two areas: isolate variation in aflatoxin production and drought stress exacerbating aflatoxin production by A. flavus. Therefore, we developed two reference pseudomolecule genome assemblies derived from chromosome arms for two isolates: AF13, a MAT1-2, highly stress tolerant, and highly aflatoxigenic isolate; and NRRL3357, a MAT1-1, less stress tolerant, and moderate aflatoxin producer in comparison to AF13. Here, we report these two reference-grade assemblies for these isolates through a combination of PacBio long-read sequencing and optical mapping, and coupled them with comparative, functional, and phylogenetic analyses. This analysis resulted in the identification of 153 and 45 unique genes in AF13 and NRRL3357, respectively. We also confirmed the presence of a unique 310 Kb insertion in AF13 containing 60 genes. Analysis of this insertion revealed the presence of a bZIP transcription factor, named atfC, which may contribute to isolate pathogenicity and stress tolerance. Phylogenomic analyses comparing these and other available assemblies also suggest that the species complex of A. flavus is polyphyletic.

The interaction between Aspergillus flavus and Zea mays is complex, and the identification of plant genes and pathways conferring resistance to the fungus has been challenging. Therefore, the authors undertook a systems biology approach involving dual RNA-seq to determine the simultaneous response from the host and the pathogen. What was dramatically highlighted in the analysis is the uniformity in the development patterns of gene expression of the host and the pathogen during infection. This led to the development of a "stage of infection index" that was subsequently used to categorize the samples before down-stream system biology analysis. Additionally, we were able to ascertain that key maize genes in pathways such as the jasmonate, ethylene and ROS pathways, were up-regulated in the study. The stage of infection index used for the transcriptomic analysis revealed that A. flavus produces a relatively limited number of transcripts during the early stages (0 to 12 h) of infection. At later stages, in A. flavus, transcripts and pathways involved in endosomal transport, aflatoxin production, and carbohydrate metabolism were up-regulated. Multiple WRKY genes targeting the activation of the resistance pathways (i.e., jasmonate, phenylpropanoid, and ethylene) were detected using causal inference analysis. This analysis also revealed, for the first time, the activation of Z. mays resistance genes influencing the expression of specific A. flavus genes. Our results show that A. flavus seems to be reacting to a hostile environment resulting from the activation of resistance pathways in Z. mays. This study revealed the dynamic nature of the interaction between the two organisms.

In an effort to control aflatoxin contamination in food and/or feed grains, a segment of research has focused on host resistance to eliminate aflatoxin from susceptible crops, including maize. To this end, screening tools are key to identifying resistant maize genotypes. The traditional field screening techniques, the kernel screening laboratory assay (KSA), and analytical methods (e.g., ELISA) used for evaluating corn lines for resistance to fungal invasion, all ultimately require sample destruction. A technological advancement on the basic BGYF presumptive screening test, fluorescence hyperspectral imaging offers an option for non-destructive and rapid image-based screening. The present study aimed to differentiate fluorescence spectral signatures of representative resistant and susceptible corn hybrids infected by a toxigenic (SRRC-AF13) and an atoxigenic (SRRC-AF36) strain of Aspergillus flavus, at several time points (5, 7, 10, and 14 days), in order to evaluate fluorescence hyperspectral imaging as a viable technique for early, non-invasive aflatoxin screening in resistant and susceptible corn lines. The study utilized the KSA to promote fungal growth and aflatoxin production in corn kernels inoculated under laboratory conditions and to provide actual aflatoxin values to relate with the imaging data. Each time point consisted of 78 kernels divided into four groups (30-susceptible, 30-resistant, 9-susceptible control, and 9-resistant control), per inoculum. On specified days, kernels were removed from the incubator and dried at 60°C to terminate fungal growth. Dry kernels were imaged with a VNIR hyperspectral sensor (image spectral range of 400–1000 nm), under UV excitation centered at 365 nm. Following imaging, kernels were submitted for the chemical AflaTest assay (VICAM). Fluorescence emissions were compared for all samples over 14 days. Analysis of strain differences separating the fluorescence emission peaks of resistant from the susceptible strain indicated that the emission peaks of the resistant strain and the susceptible strains differed significantly (p < 0.01) from each other, and there was a significant difference in fluorescence intensity between the treated and control kernels of both strains. These results indicate a viable role of fluorescence hyperspectral imaging for non-invasive screening of maize lines with divergent resistance to invasion by aflatoxigenic fungi.

Aflatoxins, highly toxic and carcinogenic to humans, are synthesized via multiple intermediates by a complex pathway in several Aspergilli, including Aspergillus flavus. Few analytical methods are available for monitoring the changes in metabolite profiles of the aflatoxin biosynthesis pathway under different growth and environmental conditions. In the present study, we developed by a D-optimal mixture design a solvent system, methanol/dichloromethane/ethyl acetate/formic acid (0.36/0.31/0.32/0.01), that was suitable for extracting the pathway metabolites. The matrix effect from dilution of cell extracts was negligible. To facilitate the identification of these metabolites, we constructed a fragmentation ion library. We further employed liquid chromatography coupled with high-resolution mass spectroscopy (UHPLC-HRMS) for simultaneous quantification of the metabolites. The limit of detection (LOD) and limit of quantitation (LOQ) were 0.002-0.016 and 0.008-0.05 μg/kg, respectively. The spiked recovery rates ranged from 81.3 to 100.3% with intraday and interday precision less than 7.6%. Using the method developed to investigate the time-course aflatoxin biosynthesis, we found that precursors, including several possible toxins (with a carcinogenic group similar to aflatoxin B1), occurred together with aflatoxin, and that production increased rapidly at the early growth stage, peaked on day four, and then decreased substantially. The maximum production of aflatoxin B1 and aflatoxin B2 occurred 1 day later. Moreover, the dominant branch pathway was the one for aflatoxin B1 formation. We revealed that the antiaflatoxigenicity mechanism of Leclercia adecarboxylata WT16 was associated with a factor upstream of the aflatoxin biosynthesis pathway. The design strategies can be applied to characterize or detect other secondary metabolites to provide a snapshot of the dynamic changes during their biosynthesis.

A comparative transcriptome analysis was performed using the genes significantly differentially expressed in cotton, corn and peanut in response to aflatoxin producing fungus Aspergillus flavus with an objective of identifying candidate resistance genes in cotton. Two-way analyses identified 732 unique genes to be differentially regulated by the fungus with only 26 genes common across all three crops that were considered candidate A. flavus resistance genes with an assumption that these genes have specific roles in conferring the resistance trait. Genes of membrane cellular component involved in DNA binding with involvement in defense responses were highly represented among the differentially expressed unique genes. Most (six) of these genes coded for 2-oxoglutarate (2OG) and Fe(II)-dependent oxygenase superfamily proteins. Genes encoding helix loop helix protein, alcohol dehydrogenase and UDP glycosylation transferase which were upregulated in response to both atoxigenic and toxigenic strains of A. flavus, could be potential resistance candidate genes for downstream functional manipulation to confer resistance.

Worldwide recognition that aflatoxin contamination of agricultural commodities by the fungus Aspergillus flavus is a global problem has significantly benefitted from global collaboration for understanding the contaminating fungus, as well as for developing and implementing solutions against the contamination. The effort to address this serious food and feed safety issue has led to a detailed understanding of the taxonomy, ecology, physiology, genomics and evolution of A. flavus, as well as strategies to reduce or control pre-harvest aflatoxin contamination, including (1) biological control, using atoxigenic aspergilli, (2) proteomic and genomic analyses for identifying resistance factors in maize as potential breeding markers to enable development of resistant maize lines, and (3) enhancing host-resistance by bioengineering of susceptible crops, such as cotton, maize, peanut and tree nuts. A post-harvest measure to prevent the occurrence of aflatoxin contamination in storage is also an important component for reducing exposure of populations worldwide to aflatoxins in food and feed supplies. The effect of environmental changes on aflatoxin contamination levels has recently become an important aspect for study to anticipate future contamination levels. The ability of A. flavus to produce dozens of secondary metabolites, in addition to aflatoxins, has created a new avenue of research for understanding the role these metabolites play in the survival and biodiversity of this fungus. The understanding of A. flavus, the aflatoxin contamination problem, and control measures to prevent the contamination has become a unique example for an integrated approach to safeguard global food and feed safety.

Summary Aflatoxin contamination in peanuts poses major challenges for vulnerable populations of sub‐Saharan Africa and South Asia. Developing peanut varieties to combat preharvest Aspergillus flavus infection and resulting aflatoxin contamination has thus far remained a major challenge, confounded by highly complex peanut–Aspergilli pathosystem. Our study reports achieving a high level of resistance in peanut by overexpressing (OE) antifungal plant defensins MsDef1 and MtDef4.2, and through host‐induced gene silencing (HIGS) of aflM and aflP genes from the aflatoxin biosynthetic pathway. While the former improves genetic resistance to A. flavus infection, the latter inhibits aflatoxin production in the event of infection providing durable resistance against different Aspergillus flavus morphotypes and negligible aflatoxin content in several peanut events/lines well. A strong positive correlation was observed between aflatoxin accumulation and decline in transcription of the aflatoxin biosynthetic pathway genes in both OE‐Def and HIGS lines. Transcriptomic signatures in the resistant lines revealed key mechanisms such as regulation of aflatoxin synthesis, its packaging and export control, besides the role of reactive oxygen species‐scavenging enzymes that render enhanced protection in the OE and HIGS lines. This is the first study to demonstrate highly effective biotechnological strategies for successfully generating peanuts that are near‐immune to aflatoxin contamination, offering a panacea for serious food safety, health and trade issues in the semi‐arid regions.

Mycotoxins are the foremost naturally occurring contaminants of food products such as corn, peanuts, tree nuts, and wheat. As the secondary metabolites, mycotoxins are mainly synthesized by many species of the genera Aspergillus, Fusarium and Penicillium, and are considered highly toxic and carcinogenic to humans and animals. Most mycotoxins are detected and quantified by analytical chemistry-based methods. While mycotoxigenic fungi are usually identified and quantified by biological methods. However, these methods are time-consuming, laborious, costly, and inconsistent because of the variability of the grain-sampling process. It is desirable to develop rapid, non-destructive and efficient methods that objectively measure and evaluate mycotoxins and mycotoxigenic fungi in food. In recent years, some spectroscopy-based technologies such as hyperspectral imaging (HSI), Raman spectroscopy, and Fourier transform infrared spectroscopy have been extensively investigated for their potential use as tools for the detection, classification, and sorting of mycotoxins and toxigenic fungal contaminants in food. HSI integrates both spatial and spectral information for every pixel in an image, making it suitable for rapid detection of large quantities of samples and more heterogeneous samples and for in-line sorting in the food industry. In order to track the latest research developments in HSI, this paper gives a brief overview of the theories and fundamentals behind the technology and discusses its applications in the field of rapid detection and sorting of mycotoxins and toxigenic fungi in food products. Additionally, advantages and disadvantages of HSI are compared, and its potential use in commercial applications is reported.

Effects of Aspergillus flavus colonization of maize kernels under different water activities (aw; 0.99 and 0.91) and temperatures (30, 37 °C) on (a) aflatoxin B1 (AFB1) production and (b) the transcriptome using RNAseq were examined. There was no significant difference (p = 0.05) in AFB1 production at 30 and 37 °C and 0.99 aw. However, there was a significant (p = 0.05) increase in AFB1 at 0.91 aw at 37 °C when compared with 30 °C/0.99 aw. Environmental stress effects using gene ontology enrichment analysis of the RNA-seq results for increasing temperature at 0.99 and 0.91 aw showed differential expression of 2224 and 481 genes, respectively. With decreasing water availability, 4307 were affected at 30 °C and 702 genes at 37 °C. Increasing temperature from 30 to 37 °C at both aw levels resulted in 12 biological processes being upregulated and 9 significantly downregulated. Decreasing aw at both temperatures resulted in 22 biological processes significantly upregulated and 25 downregulated. The interacting environmental factors influenced functioning of the secondary metabolite gene clusters for aflatoxins and cyclopiazonic acid (CPA). An elevated number of genes were co-regulated by both aw and temperature. An interaction effect for 4 of the 25 AFB1 genes, including regulatory and transcription activators occurred. For CPA, all 5 biosynthetic genes were affected by aw stress, regardless of temperature. The molecular regulation of A. flavus in maize is discussed.

Screening of gene manipulation events (transgenic, mutation/genome editing, etc.) is a cost/labor-intensive and time-consuming process in plant science research. While polymerase chain reaction (PCR) is the most commonly used method for screening, the process still requires efficient DNA extraction and subsequent confirmation. However, PCR cannot predict gene expression. To screen a larger number of transgenic plants, it would be ideal to develop a quick and reliable screening procedure. We have applied a Liberty® leaf-painting method (against bar gene under 4x35S promoter) to screen transgenic maize (Zea mays L.) plants and validated the results through PCR and quantitative real-time PCR (qRT-PCR). Liberty leaf painting at 500 mg L−1a.i. was > 95% accurate in identifying transgenic events that agreed with the PCR results. Further investigation of bar gene expression in sensitive lines that were PCR positive shows very low expression of the bar gene. We have provided a simple, and rapid assay to d...

Xaa-Pro dipeptidase (XPD) catalyzes hydrolysis of iminopeptide bond in dipeptides containing trans-proline as a second residue. XPDs are found in all living organisms and are believed to play an essential role in proline metabolism. Here, we report crystal structures and extensive enzymatic studies of XPD from Xanthomonas campestris (XPDxc), the first such comprehensive study of a bacterial XPD. We also report enzymatic activities of its ortholog from Mycobacterium tuberculosis (XPDmt). These enzymes are strictly dipeptidases with broad substrate specificities. They exhibit substrate inhibition and allostericity, as described earlier for XPD from Lactococcus lactis (XPDll). The structural, mutational and comparative data have revealed a novel mechanism of dipeptide selectivity and substrate binding in these enzymes. Moreover, we have identified conserved sequence motifs that distinguish these enzymes from other prolidases, thus defining a new subfamily. This study provides a suitable structural template for explaining unique properties of this XPDxc subfamily. In addition, we report unique structural features of XPDxc protein like an extended N-terminal tail region and absence of a conserved Tyr residue near the active site.

Aspergillus flavus infects several food and feed crops, such as corn (Zea mays L.), cotton (Gossypium hirsutum L.), peanuts (Arachis hypogaea L.), and tree nut crops and contaminates the seed with carcinogenic aflatoxins. These susceptible crops contain rich reserves of lipids and fatty acids. The nature of relationship between lipids and the ability of the fungus to infect and produce aflatoxins in mature cottonseed, a protein-rich animal feed, has been addressed previously. In this study, we tracked lipid accumulation in developing cottonseed (15–35 days post-anthesis [DPA]) and also the ability of an aflatoxigenic strain and an isogenic non-aflatoxigenic strain to grow and produce aflatoxins in planta. The aflatoxigenic strain Af-70 green fluorescent protein (GFP) and the isogenic, non-aflatoxigenic strain SRRC 1500 (fungal collections maintained at the Southern Regional Research Center) did not differ much in infection and colonization of cottonseed. The non-aflatoxigenic strain did not produc...

The potential of near infrared (NIR) hyperspectral imaging over the 900-2500 nm spectral range was examined for discrimination of artificially-inoculated corn kernels with aflatoxigenic and non-aflatoxigenic strains of Aspergillus flavus in this study. The two A. flavus strains, aflatoxigenic AF13 and non-aflatoxigenic AF36 were used for inoculation on corn kernels. Four treatments were included, with each treatment consisting of 100 kernels. Each treatment of 100 kernels were artificially inoculated with AF13 or AF36 strain and incubated at 30 °C for 3 and 8 days, separately. The mean spectra were extracted from the collected NIR hyperspectral images for individual corn kernels, and then based on the mean spectra, the principal component analysis combined with linear discriminant analysis (PCA-LDA) method was employed to establish the classification models. The pairwise classification models were established by the PCA-LDA method to discriminate the AF36-inoculated and the AF13-inoculated kernels at different incubation days. All the overall accuracies obtained by the pairwise models were ≥98.0%. A common model that takes the AF13-inoculated kernels at different incubation days as one class and the AF36-inoculated kernels at different incubation days as the second class, achieved an overall accuracy of 99.0% for the prediction samples. This indicates a great potential of using NIR hyperspectral imaging to classify corn kernels infected by aflatoxigenic and non-aflatoxigenic A. flavus regardless of infection time.

Conventional methods for detecting aflatoxigenic fungus and aflatoxin contamination are generally time-consuming, sample-destructive, and require skilled personnel to perform, making them impossible for large-scale nondestructive screening detection, real-time, and on-site analysis. Therefore, the potential of visible-near-infrared (Vis-NIR) spectroscopy over the 400-2500 nm spectral range was examined for determination of aflatoxigenic fungus infection and the corresponding aflatoxin contamination on corn kernels in a rapid and nondestructive manner. The two A. flavus strains, AF13 and AF38, were used to represent the aflatoxigenic fungus and nonaflatoxigenic fungus, respectively, for artificial inoculation on corn kernels. The partial least-squares discriminant analysis (PLS-DA) models based on different combinations of spectral range (I: 410-1070 nm; II: 1120-2470 nm), corn side (endosperm or germ side), spectral variable number (full spectra or selected variables), modeling approach (two-step or one-step), and classification threshold (20 or 100 ppb) were developed and their performances were compared. The first study focusing on detection of aflatoxigenic fungus-infected corn kernels showed that, in classifying the "control+AF38-inoculated" and AF13-inoculated corn kernels, the full spectral PLS-DA models using the preprocessed spectra over range II and one-step approach yielded more accurate prediction results than using the spectra over range I and the two-step approach. The advantage of the full spectral PLS-DA models established using one corn side than the other side were not consistent in the explored combination cases. The best full spectral PLS-DA model obtained was obtained using the germ-side spectra over range II with the one-step approach, which achieved an overall accuracy of 91.11%. The established CARS-PLSDA models performed better than the corresponding full-spectral PLS-DA models, with the better model achieved an overall accuracy of 97.78% in separating the AF13-inoculated corn kernels and the uninfected control and AF38-inoculated corn kernels. The second study focusing on the detection of aflatoxin-contaminated corn kernels showed that, based on the aflatoxin threshold of 20 and 100 ppb, the best overall accuracy in classifying the aflatoxin-contaminated and healthy corn kernels attained 86.67% and 84.44%, respectively, using the CARS-PLSDA models. The quantitative modeling results using partial least-squares regression (PLSR) obtained the correlation coefficient of prediction set ( R

Macrophages polarize to proinflammatory M1 or anti-inflammatory M2 states with distinct physiological functions. This transition within the M1–M2 phenotypes decides the nature, duration and severity of an inflammatory response. Although there is a substantial understanding of the fate of these phenotypes, the underlying molecular mechanism of transition within the M1–M2 phenotypes is not well understood. We have investigated the role of neuronal nitric oxide synthase (NOS1)-mediated regulation of activator protein 1 (AP-1) transcription factor in macrophages as a critical effector of macrophage phenotypic change. Raw 264.7 and THP1 macrophages were stimulated with LPS (250 ng/ml) to activate the inflammatory signaling pathway. We analyzed the effect of pharmacological NOS1 inhibitor: TRIM (1-(2- Trifluoromethylphenyl) imidazole) on LPS-induced inflammatory response in macrophages. We determined that NOS1-derived nitric oxide (NO) facilitate Fos and Jun interaction which induces IL-12 & IL-23 expression. Pharmacological inhibition of NOS1 inhibits ATF2 and Jun dimer. Switching of Fos and Jun dimer to ATF2 and Jun dimerization controls phenotype transition from IL-12high IL-23high IL-10low to IL-12low IL-23lowIL-10high phenotype, respectively. These findings highlight a key role of the TLR4-NOS1-AP1 signaling axis in regulating macrophage polarization.

Aspergillus flavus is an opportunistic and pathogenic fungus that infects several crops of agricultural importance and under certain conditions may produce carcinogenic mycotoxins. Rising global temperatures, disrupted precipitation patterns and increased CO2 levels that are associated with future climate conditions are expected to impact the growth and toxigenic potential of A. flavus. Both laboratory and real world observations have demonstrated this potential, especially when examining the effects of water availability and temperature. Recent experiments have also established that CO2 may also be affecting toxin production. The application of current technologies in the field of functional genomics, including genomic sequencing, RNA-seq, microarray technologies and proteomics have revealed climate change-related, abiotic regulation of the aflatoxin cluster and influence on the plant-fungus interaction. Furthermore, elevated CO2 levels have been shown to impact expression of the aflatoxin biosynthetic regulatory gene aflR. The use of functional genomics will allow researchers to better understand the underlying transcriptomic response within the fungus to climate change, with a view towards predicting changes in fungal infection and toxin production associated with climate change.

Vitex negundo is a medicinal plant used in Indian and folklore medicines to cure various ailments including arthritis. In the present study, the hydroethanolic extract of V. negundo leaves (VNE) were evaluated for antioxidant, genoprotective and immunomodulatory activity in Freund’s complete adjuvant (FCA) induced arthritis. VNE was evaluated for in vitro antioxidant activity using various parameters including plasmid nicking assay. VNE (200 mg/kg body weight) was orally administered to FCA induced arthritic rats to evaluate its genoprotective effects on peripheral blood leukocytes using the alkaline comet assay. Furthermore, the effects of VNE treatment on serum proinflammatory mediators such as tumor necrosis factor-α (TNF-α) and interleukin -1α (IL-1α) and the hematological parameters such as total RBC count, hematocrit and hemoglobin were measured in arthritic rats. The results showed that VNE exhibited potential in vitro antioxidant and DNA protecting activities in a concentration dependent manner. The phytoconstituents of VNE showed a strong and positive correlation with the antioxidant properties. DNA damage in the peripheral blood leukocytes of arthritic animals was significantly decreased after VNE treatment. VNE showed inhibition of serum proinflammatory cytokines (TNF-α and IL-1α) however, the inhibition was statistically not-significant. The arthritic animals showed significant increase in hemolysis, which was restored by VNE treatment. The results of the present study indicate that VNE may be a source of antioxidant, genoprotective with immunomodulatory activity, which may be attributed to its antioxidant phytoconstituents.

Maize (Zea mays L.) is one of the major crops susceptible to Aspergillus flavus Link ex. Fries infection and subsequent aflatoxin contamination. Previous studies found the production of an antifungal 14 kDa trypsin inhibitor (TI) was associated with maize aflatoxin resistance. To further investigate whether the TI plays any direct role in resistance, a TI gene silencing vector was constructed and transformed into maize. Mature kernels were produced from 66 transgenic lines representing 18 independent events. A final total of twelve lines representing four independent events were confirmed positive for transformation, five of which showed significant reduction (63 to 88%) in TI transcript abundance in seedling leaf tissue and seven of which showed significant TI protein reduction (39-85%) in mature kernels. Six of the seven silenced transgenic lines supported higher levels of aflatoxin production compared to negative controls. To further confirm the role of TI in field resistance to aflatoxin accumulation, DNA sequence polymorphisms from within the gene or linked simple sequence repeats were tested in four quantitative trait loci (QTL) mapping populations for QTL effect, and three QTL with log of the odds scores of 11, 4.5, and 3.0 and possibly caused by the TI protein encoding gene were found. Sequence polymorphisms were also tested for association to aflatoxin levels in an association mapping panel, and three single nucleotide polymorphisms were found associated with aflatoxin accumulation (P

Aspergillus flavus is a plant and animal pathogen that also produces the potent carcinogen aflatoxin. Aspergillus oryzae is a closely related species that has been used for centuries in the food fermentation industry and is Generally Regarded As Safe (GRAS). Whole genome sequences for these two fungi are now complete, providing us with the opportunity to examine any genomic differences that may explain the different ecological niches of these two fungi, and perhaps to identify pathogenicity factors in A. flavus. These two fungi are very similar in genome size and number of predicted genes. The estimated genome size (36·8 Mb) and predicted number of genes (12 197) for A. flavus is similar to that of A. oryzae (36·7 Mb and 12 079, respectively). These two fungi have significantly larger genomes than Aspergillus nidulans (30·1) and Aspergillus fumigatus (29·4). The A. flavus and A. oryzae genomes are enriched in genes for secondary metabolism, but do not differ greatly from one another in the predicted number of polyketide synthases, nonribosomal peptide synthases or the number of genes coding for cytochrome P450 enzymes. A micro-scale analysis of the two fungi did show differences in DNA correspondence between the two species and in the number of transposable elements. Each species has approximately 350 unique genes. The high degree of sequence similarity between the two fungi suggests that they may be ecotypes of the same species and that A. oryzae has resulted from the domestication of A. flavus.

Background Hemidesmus indicus is an important medicinal plant and extensively used in Ayurvedic and Unani system of medicine. The aim of the study was to evaluate the antioxidant, hypoglycaemic and hypolipidemic potential of methanolic extract of roots of Hemidesmus indicus (HIE) in streptozotocin (STZ) induced diabetic mice. Methods HIE was analyzed by LC-MS to determine its phytochemical composition. The in-vitro antioxidant activity of HIE was analyzed through inhibition of free radical scavenging activity (FRSA), total antioxidant power (TAP) and reducing power. Diabetes in mice was induced by a single dose of STZ followed by HIE treatment. The antioxidative, hypoglycaemic and hypolipidemic activity were studied ex-vivo in tissues of diabetic mice. Results Phytochemical composition of hemidesmus indicus roots (HIE) revealed the presence of phenols, flavanoids, terpenoids and about 40 different phytoconstituents by LC-MS analysis. Inhibition of lipid peroxidation (LPO) and modulation in superoxide dismutase (SOD), catalase (CAT), glutathione-S- transferase (GST) activity and glutathione (GSH) content showed potent antioxidant activity of HIE in STZ induced diabetic mice, which was also substantiated by in-vitro antioxidant assays. The decrease in fasting blood glucose and serum lipid profile was also observed in mice administered HIE. Conclusion It is proposed that HIE modulates the oxidant/antioxidant in favor of reducing oxidative stress, hypoglycemia and improved the lipid profile in treated groups.

Aspergillus flavus is a saprophytic fungus that infects corn, peanuts, tree nuts and other agriculturally important crops. Once the crop is infected the fungus has the potential to secrete one or more mycotoxins, the most carcinogenic of which is aflatoxin. Aflatoxin contaminated crops are deemed unfit for human or animal consumption, which results in both food and economic losses. Within A. flavus, two morphotypes exist: the S strains (small sclerotia) and L strains (large sclerotia). Significant morphological and physiological differences exist between the two morphotypes. For example, the S-morphotypes produces sclerotia that are smaller (< 400 μm), greater in quantity, and contain higher concentrations of aflatoxin than the L-morphotypes (>400 μm). The morphotypes also differ in pigmentation, pH homeostasis in culture and the number of spores produced. Here we report the first full genome sequence of an A. flavus S morphotype, strain AF70. We provide a comprehensive comparison of the A. flavus S-morphotype genome sequence with a previously sequenced genome of an L-morphotype strain (NRRL 3357), including an in-depth analysis of secondary metabolic clusters and the identification SNPs within their aflatoxin gene clusters.

Maize (Zea mays L.) is one of the major crops susceptible to Aspergillus flavus infection and subsequent aflatoxin contamination. Many earlier studies indicated the roles of kernel proteins, especially constitutively expressed proteins, in maize resistance to A. flavus infection and aflatoxin production. In this review, we examined the past and current efforts in identifying maize genes and proteins from kernel, rachis, and silk tissues that may play an important role in resistance to A. flavus infection and aflatoxin contamination, as well as the efforts in determining the importance or involvement of them in maize resistance through biochemical, molecular and genetics studies. Through these studies, we gained a better understanding of host resistance mechanism: resistant lines appear to either express some stress-related and antifungal proteins at higher levels in endosperm, embryo, rachis and silk tissues before A. flavus infection or induce the expression of these proteins much faster compared to susceptible maize lines. In addition, we summarised several recent efforts in enhancing maize resistance to aflatoxin contamination using native genes from maize or heterologous and synthetic genes from other sources as well as from A. flavus. These efforts to either suppress A. flavus growth or aflatoxin production, have all shown some promising preliminary success. For example, maize plants transformed with an ?-amylase inhibitor protein from Lablab purpurea showed reduced aflatoxin levels by 56% in kernel screening assays. The antifungal potentials of transgenic maize plants expressing synthetic lytic peptides, such as cecropin-based D4E1 or tachyplesin-based AGM peptides with demonstrated anti-flavus activity (IC50 = 2.5 to 10 ?M), are yet to be assayed. Further investigation in these areas may provide a more cost-effective alternative to biocontrol in managing aflatoxin contamination in maize and other susceptible crops.

Competition with Aspergillus flavus isolates incapable of aflatoxin production is currently the most widely used biocontrol method for reducing aflatoxin contamination in maize and cottonseed where aflatoxin contamination is a persistent problem for human and animal health. The method involves spreading non-aflatoxigenic A. flavus spores onto the field prior to harvest. How competition works is not fully understood. Current theories suggest that atoxigenic A. flavus either simply displaces aflatoxin-producing isolates or that competition is an active inhibition process that occurs when the fungi occupy the same locus on the plant. In this paper we describe several challenges that the biocontrol strategy should address before this practice is introduced worldwide. These include the need to better understand the diversity of A. flavus populations in the agricultural soil, the effects of climate change on both this diversity and on plant susceptibility, the ability of the introduced biocontrol strain to outcross with existing aflatoxin-producing A. flavus, the adaptation of certain A. flavus isolates for predominant growth on the plant rather than in the soil, the difficulty in timing the application or controlling the stability of the inoculum, the effect of the introduction of the biocontrol strain on the soil microenvironment, the potential damage to the plant from the introduced strain, and the overall need to better understand the entire A. flavus toxin burden, beyond that of aflatoxin, that may result from A. flavus contamination. In addition, the cost/benefit ratio for the biocontrol method should be considered in comparing this method to other methods for reducing food and feed contamination with aflatoxins.

Aspergillus flavus is a saprophytic fungus that may colonize several important crops, including cotton, maize, peanuts and tree nuts. Concomitant with A. flavus colonization is its potential to secrete mycotoxins, of which the most prominent is aflatoxin. Temperature, water activity (aw) and carbon dioxide (CO2) are three environmental factors shown to influence the fungus-plant interaction, which are predicted to undergo significant changes in the next century. In this study, we used RNA sequencing to better understand the transcriptomic response of the fungus to aw, temperature, and elevated CO2 levels. We demonstrate that aflatoxin (AFB1) production on maize grain was altered by water availability, temperature and CO2. RNA-Sequencing data indicated that several genes, and in particular those involved in the biosynthesis of secondary metabolites, exhibit different responses to water availability or temperature stress depending on the atmospheric CO2 content. Other gene categories affected by CO2 levels alone (350 ppm vs. 1000 ppm at 30 °C/0.99 aw), included amino acid metabolism and folate biosynthesis. Finally, we identified two gene networks significantly influenced by changes in CO2 levels that contain several genes related to cellular replication and transcription. These results demonstrate that changes in atmospheric CO2 under climate change scenarios greatly influences the response of A. flavus to water and temperature when colonizing maize grain.

Aspergillus flavus is an opportunistic plant pathogen that colonizes and produces the toxic and carcinogenic secondary metabolites, aflatoxins, in oil-rich crops such as maize (Zea mays ssp. mays L.). Pathogenesis-related proteins serve as an important defense mechanism against invading pathogens by conferring systemic acquired resistance (SAR) in plants. Among these, production of the pathogenesis-related maize seed protein, ZmPRms (AC205274.3_FG001), has been speculated to be involved in resistance to infection by A. flavus and other pathogens. To better understand the relative contribution of ZmPRms to A. flavus resistance and aflatoxin production, a seed-specific RNA interference (RNAi)-based gene silencing approach was used to develop transgenic maize lines expressing hairpin RNAs to target ZmPRms. Down-regulation of ZmPRms in transgenic kernels resulted in a ~250-350% increase in A. flavus infection accompanied by a ~4.5 to 7.5-fold higher accumulation of aflatoxins than control plants. Gene co-expression network analysis of RNA-seq data during the A. flavus-maize interaction identified ZmPRms as a network hub possibly responsible for regulating several downstream candidate genes associated with disease resistance and other biochemical functions. Expression analysis of these candidate genes in the ZmPRms-RNAi lines demonstrated down-regulation (vs. control) of a majority of these ZmPRms regulated genes during A. flavus infection. These results are consistent with a key role of ZmPRms in resistance to A. flavus infection and aflatoxin accumulation in maize kernels.

Non-invasive, easy to use and cost-effective technology offers a valuable alternative for rapid detection of carcinogenic fungal metabolites, namely aflatoxins, in commodities. One relatively recent development in this area is the use of spectral technology. Fluorescence hyperspectral imaging, in particular, offers a potential rapid and non-invasive method for detecting the presence of aflatoxins in maize infected with the toxigenic fungus Aspergillus flavus. Earlier studies have shown that whole maize kernels contaminated with aflatoxins exhibit different spectral signatures from uncontaminated kernels based on the external fluorescence emission of the whole kernels. Here, the effect of time on the internal fluorescence spectral emissions from cross-sections of kernels infected with toxigenic and atoxigenic A. flavus, were examined in order to elucidate the interaction between the fluorescence signals emitted by some aflatoxin contaminated maize kernels and the fungal invasion resulting in the production of aflatoxins. First, the difference in internal fluorescence emissions between cross-sections of kernels incubated in toxigenic and atoxigenic inoculum was assessed. Kernels were inoculated with each strain for 5, 7, and 9 days before cross-sectioning and imaging. There were 270 kernels (540 halves) imaged, including controls. Second, in a different set of kernels (15 kernels/group; 135 total), the germ of each kernel was separated from the endosperm to determine the major areas of aflatoxin accumulation and progression over nine growth days. Kernels were inoculated with toxigenic and atoxigenic fungal strains for 5, 7, and 9 days before the endosperm and germ were separated, followed by fluorescence hyperspectral imaging and chemical aflatoxin determination. A marked difference in fluorescence intensity was shown between the toxigenic and atoxigenic strains on day nine post-inoculation, which may be a useful indicator of the location of aflatoxin contamination. This finding suggests that both, the fluorescence peak shift and intensity as well as timing, may be essential in distinguishing toxigenic and atoxigenic fungi based on spectral features. Results also reveal a possible preferential difference in the internal colonization of maize kernels between the toxigenic and atoxigenic strains of A. flavus suggesting a potential window for differentiating the strains based on fluorescence spectra at specific time points.

Aflatoxin contamination in peanut products has been an important and long-standing problem around the world. Produced mainly by Aspergillus flavus and Aspergillus parasiticus, aflatoxins are the most toxic and carcinogenic compounds among toxins. This study investigated the application of fluorescence visible near-infrared (VNIR) hyperspectral images to assess the spectral difference between peanut kernels inoculated with toxigenic and atoxigenic inocula of A. flavus and healthy kernels. Peanut kernels were inoculated with NRRL3357, a toxigenic strain of A. flavus, and AF36, an atoxigenic strain of A. flavus, respectively. Fluorescence hyperspectral images under ultraviolet (UV) excitation were recorded on peanut kernels with and without skin. Contaminated kernels exhibited different fluorescence features compared with healthy kernels. For the kernels without skin, the inoculated kernels had a fluorescence peaks shifted to longer wavelengths with lower intensity than healthy kernels. In addition, the fluorescence intensity of peanuts without skin was higher than that of peanuts with skin (10 times). The fluorescence spectra of kernels with skin are significantly different from that of the control group (p

Acylpeptide hydrolase (APH; EC 3.4.19.1), which belongs to the S9 family of serine peptidases (MEROPS), catalyzes the removal of anN-acylated amino acid from a blocked peptide. The role of this enzyme in mammalian cells has been suggested to be in the clearance of oxidatively damaged proteins as well as in the degradation of the β-amyloid peptides implicated in Alzheimer's disease. Detailed structural information for the enzyme has been reported from two thermophilic archaea; both of the archaeal APHs share a similar monomeric structure. However, the mechanisms of substrate selectivity and active-site accessibility are totally different and are determined by inter-domain flexibility or the oligomeric structure. An APH homologue from a bacterium,Deinococcus radiodurans(APHdr), has been crystallized using microbatch-under-oil employing the random microseed matrix screening method. The protein crystallized in space groupP21, with unit-cell parametersa= 77.6,b= 189.6,c= 120.4 Å, β = 108.4°. A Matthews coefficient of 2.89 Å3 Da−1corresponds to four monomers, each with a molecular mass of ∼73 kDa, in the asymmetric unit. The APHdr structure will reveal the mechanisms of substrate selectivity and active-site accessibility in the bacterial enzyme. It will also be helpful in elucidating the functional role of this enzyme inD. radiodurans.

The persistent occurrence of aflatoxins in food and feed remains a problem for producers of commodities subject to colonization with toxigenic molds. Aflatoxins are secondary metabolites of fungi of the Aspergillus spp. associated with deleterious health effects. Because current screening methods for these toxins are lengthy, destructive, and costly, there is a continuous search for a more rapid, noninvasive, and cost-effective technology. The present study utilized a fluorescence excitation–emission matrix (EEM) of aflatoxin as well as two additional secondary metabolites (kojic acid and the bright greenish-yellow fluorescence (BGYF) compound) of Aspergillus flavus measured with a fluorescence spectrophotometer. The results were compared to image data acquired with a fluorescence hyperspectral sensor in order to evaluate the potential of image-based technology for detecting aflatoxin in grain. The excitation–emission matrix of aflatoxin B1 standard produced overlapping peaks in 340–400 nm of excitation range emitting in the blue range at around 450 nm. The spectral signature extracted from the hyperspectral image was also in the blue range, emitting blue fluorescence. Because the results from both systems were comparable, where all fluorescence peaks were in the blue range, the present study validates the feasibility of image-based technology for nondestructive detection of aflatoxin in corn. Additional peaks were revealed in the aflatoxin EEM in the 260-nm excitation range that were not present in the kojic acid and BGYF compound mixture. This new information allows for the separation of the aflatoxin signature from the potentially confounding overlap of other secondary metabolites occurring in the blue and blue-green spectral ranges.

Aflatoxins are toxic secondary metabolites predominantly produced by the fungi Aspergillus flavus and Aspergillus parasiticus. Aflatoxin-contaminated corn is toxic to domestic animals when ingested in feed and is a known carcinogen associated with liver and lung cancer in humans. Consequently, aflatoxin levels in food and feed are regulated by the U.S. Food and Drug Administration (FDA), which allows 20 ppb (parts per billion) limits in food and 100 ppb in feed for interstate commerce. Currently, aflatoxin detection and quantification methods are based on analytical tests, including thin-layer chromatography (TCL) and high-performance liquid chromatography (HPLC). These analytical tests require the destruction of samples and are costly and time consuming. Thus, the ability to detect aflatoxin in a rapid, non-destructive way is crucial to the grain industry, particularly the corn industry. Hyperspectral imaging technology offers a non-invasive approach to screening for food safety inspection and quality control based on spectral signatures. The focus of this study was to classify aflatoxin-contaminated single corn kernels using fluorescence hyperspectral imagery. Field-inoculated corn kernels were used in the study. Contaminated and control kernels under long-wavelength ultraviolet excitation were imaged using a visible near-infrared (VNIR) hyperspectral camera. The imaged kernels were chemically analyzed to provide reference information for image analysis. This article describes a procedure for processing corn kernels located in different images for statistical training and classification. Two classification algorithms (maximum likelihood and binary encoding) were used to classify each corn kernel as “control” or “contaminated” through pixel classification. The binary encoding approach had a slightly better performance, with accuracy equal to 87% or 88% when 20 ppb or 100 ppb, respectively, was used as the classification threshold. In addition, three narrow-band fluorescence indices were developed and tested in this study. It was found that the highest correlation was -0.81 with the normalized difference fluorescence index (NDFI). The two bands used for the NDFI were 437 and 537 nm. The use of key wavelengths for contamination detection would be helpful for developing rapid and non-invasive inspection systems. This study demonstrated the potential of using fluorescence hyperspectral imagery for aflatoxin contamination detection in corn kernels infected with A. flavus.

To study the possible role of microwave (MW) exposure on spatial memory of Swiss albino mice and its relationship to protein concentration in whole brain.Mice were exposed to 10 GHz (Giga Hertz) microwaves with the power density of 0.25 mW/cm(2) (milliwatt per centimeter square) with average whole body specific absorption rate (SAR) 0.1790 W/kg daily for 2 hours per day (h/day) for 30 days. After exposure mice were tested for spatial memory performance using Morris water maze test (MWT). For this purpose mice (6-8 weeks old) were divided into two groups: (i) Sham exposed, and (ii) microwaves exposed. After initial training for two days, MWT was performed for another 6 days. Protein was estimated 48 h after exposure and immediately after completion of MWT.Both sham-exposed and microwaves-exposed animals showed a significant decrease in escape time with training. Microwaves-exposed animals had statistically significant higher mean latency to reach the target quadrant compared to sham exposed. A concurrent decrease in protein levels was estimated in whole brain of the exposed mice compared to sham-exposed mice.It can be concluded from the current study that exposure to microwave radiation caused decrements in the ability of mice to learn the special memory task, this may be due to simultaneous decrease in protein levels in the brain of mice.

In an effort to address the problem of rapid detection of aflatoxin in grain, particularly oilseeds, the current study assessed the spectral differences of aflatoxin production in kernels from a cornfield inoculated with spores from 2 different strains of toxigenic Aspergillus flavus. Aflatoxin production in corn from the same field due to natural infestation was also assessed. A small corn plot in Baton Rouge, La., U.S.A., was used during the 2008-growing season. Two groups of 400 plants were inoculated with 2 different inocula and 1 group of 400 plants was designated as controls. Any contamination detected in the controls was attributed to natural infestation. A subset of each group was imaged with a visible near infra red (VNIR) hyperspectral system under ultra violet (UV) excitation and subsequently analyzed for aflatoxin using affinity column fluorometry. Group differences were statistically analyzed. Results indicate that when all the spectral data across all groups were averaged, any potential differences between groups (treated and untreated) were obscured. However, spectral analysis based on contaminated "hot" pixel classification showed a distinct spectral shift/separation between contaminated and clean ears with fluorescence peaks at 501 and 478 nm, respectively. All inoculated and naturally infected control ears had fluorescence peaks at 501 nm that differed from uninfected corn ears. Results from this study may be useful in evaluating rapid, noninvasive instrumentation and/or methodology for aflatoxin detection in grain.

Aflatoxin contamination caused by Aspergillus flavus infection of corn is a significant and chronic threat to corn being used as food or feed. Contamination of crops at levels of 20 ng g(-1) or higher (as regulated by the USFDA) by this toxin and potent carcinogen makes the crop unsalable, resulting in a significant economic burden on the producer. This review focuses on elimination of this contamination in corn which is a major US crop and the basis of many products. Corn is also "nature's example" of a crop containing heritable resistance to aflatoxin contamination, thereby serving as a model for achieving resistance to aflatoxin contamination in other crops as well. This crop is the largest production grain crop worldwide, providing food for billions of people and livestock and critical feedstock for production of biofuels. In 2011, the economic value of the US corn crop was US$76 billion, with US growers producing an estimated 12 billion bushels, more than one-third of the world's supply. Thus, the economics and significance of corn as a food crop and the threat to food safety due to aflatoxin contamination of this major food crop have prompted the many research efforts in many parts of the world to identify resistance in corn to aflatoxin contamination. Plant breeding and varietal selection has been used as a tool to develop varieties resistance to disease. This methodology has been employed in defining a few corn lines that show resistance to A. flavus invasion; however, no commercial lines have been marketed. With the new tools of proteomics and genomics, identification of resistance mechanisms, and rapid resistance marker selection methodologies, there is an increasing possibility of finding significant resistance in corn, and in understanding the mechanism of this resistance.

Trans-2-hexenal (T2H), a plant-produced aldehyde, was intermittently pumped over a 7 d period into a small, bench top model of stored corn (nonsterile, moisture content about 23%). Naturally occurring bacteria and fungi, including added Aspergillus flavus, grew rapidly on corn not treated with T2H vapor. However, intermittently pumped T2H (30 min per 2 h or 30 min per 12 h) significantly reduced bacterial and fungal viable populations, with nearly 100% fungal viability loss observed after either (1) one day of pumping at the 30 min per 2 h rate or (2) pumping cycles of 30 min per 12 h period over the initial 48 to 72 h of incubation. Data suggest that short-term intermittent fumigation of stored corn with T2H could prevent growth of bacteria and mycotoxigenic fungi such as A. flavus. Practical Application Intermittent pumping of volatile T2H for as little as 30 min per 12 h period over the initial 72 h could prevent the growth of naturally occurring bacteria and fungi, especially mycotoxigenic fungi, on stored, nonsterile high moisture corn.

Resistance or susceptibility of maize inbreds to infection by Aspergillus flavus was evaluated by the kernel screening assay. A green fluorescent protein-expressing strain of A. flavus was used to measure fungal spread and aflatoxin levels in real-time following fungal infection of kernels. Among the four inbreds tested, MI82 showed the most resistance and Ga209 the least. TZAR101 was also resistant to fungal infection, whereas Va35 was susceptible to fungal infection. However, Va35 produced lower aflatoxin levels compared to the susceptible line Ga209. Fluorescence microscopy indicated that the site of entry of the fungus into the kernel was consistently through the pedicel. Entry through the pericarp was never observed in undamaged kernels. In view of these results, incorporation or overexpression of antifungal proteins should be targeted to the pedicel and basal endosperm region in developing kernels. Once the fungus has entered through the pedicel, it spreads quickly through the open spaces between the pericarp and the aleurone layer, ultimately colonising the endosperm and scutellum and, finally, the embryo. A clear correlation was established between fungal fluorescence and aflatoxin levels. This method provides a quick, reliable means of evaluating resistance to A. flavus in undamaged kernels and provides breeders with a rapid method to evaluate maize germplasm.

A gene co-expression network was generated using a dual RNA-seq study with the fungal pathogen A. flavus and its plant host Z. mays during the initial 3 days of infection. The analysis deciphered novel pathways and mapped genes of interest in both organisms during the infection. This network revealed a high degree of connectivity in many of the previously recognized pathways in Z. mays such as jasmonic acid, ethylene, and reactive oxygen species (ROS). For the pathogen A. flavus, a link between aflatoxin production and vesicular transport was identified within the network. There was significant interspecies correlation of expression between Z. mays and A. flavus for a subset of 104 Z. mays, and 1942 A. flavus genes. This resulted in an interspecies subnetwork enriched in multiple Z. mays genes involved in the production of reactive oxygen species. In addition to the ROS from Z. mays, there was enrichment in the vesicular transport pathways and the aflatoxin pathway for A. flavus. Included in these genes, a key aflatoxin cluster regulator, AflS, was found to be co-regulated with multiple Z. mays ROS producing genes within the network, suggesting AflS may be monitoring host ROS levels. The entire gene expression network for both host and pathogen, and the subset of interspecies correlations, is presented as a tool for hypothesis generation and discovery for events in the early stages of fungal infection of Z. mays by A. flavus.

Aflatoxins are the most potent naturally occurring carcinogens of fungal origin. Biosynthesis of aflatoxin involves the coordinated expression of more than 25 genes. The function of one gene in the aflatoxin gene cluster, aflJ, is not entirely understood but, because previous studies demonstrated a physical interaction between the Zn2Cys6 transcription factor AflR and AflJ, AflJ was proposed to act as a transcriptional co-activator. Image analysis revealed that, in the absence of aflJ in A. parasiticus, endosomes cluster within cells and near septa. AflJ fused to yellow fluorescent protein complemented the mutation in A. parasiticus ΔaflJ and localized mainly in endosomes. We found that AflJ co-localizes with AflR both in endosomes and in nuclei. Chromatin immunoprecipitation did not detect AflJ binding at known AflR DNA recognition sites suggesting that AflJ either does not bind to these sites or binds to them transiently. Based on these data, we hypothesize that AflJ assists in AflR transport to or from the nucleus, thus controlling the availability of AflR for transcriptional activation of aflatoxin biosynthesis cluster genes. AflJ may also assist in directing endosomes to the cytoplasmic membrane for aflatoxin export.

The in vitro antioxidant potential and in vivo radioprotective ability of soy isoflavones was studied. Male Wistar rats were orally administered with soybean isoflavones (60 mg/kg) for 21 days followed by gamma irradiation exposure. Survival studies in rats exposed at 10 Gy and endogenous spleen colony forming unit assay (CFU) at 6.0 Gy were performed in order to find radioprotective and immunomodulatory nature of the compound. The rat liver post mitochondrial supernatant and erythrocytes were used to measure lipid peroxidation (LPO) and glutathione (GSH) content along with various antioxidant enzymes like superoxide dismutase (SOD), catalase (CAT) and glutathione-S-transferase (GST) after gamma irradiation exposure at 2.0 Gy. Pretreatment with soy isoflavone, prior to gamma irradiation resulted in the increased survival rate of the animals as compared to irradiated group. CFU counts in the isoflavone treated group followed by gamma irradiation at 6 Gy were significantly high as compared to control and the irradiated group, showing immunomodulatory nature of the isoflavones. Pretreatment with isoflavones also significantly reduced the LPO, enhanced the activity of antioxidant enzymes and improved haematological and histological parameters. The present study suggests that supplementation with isoflavone has potent antioxidant activity and act as probable radioprotector against gamma radiation induced oxidative damage.

The present study is an attempt to study the effects of 10 GHz (Giga Hertz) microwaves irradiation in Swiss albino mice. The animals of 6-8 weeks of age were divided into 2 groups (i) sham exposed (ii) microwaves. The mice were exposed to 10 GHz microwaves daily for 2 hours per day for 30 days. The power density was 0.25 mW/cm2 (milliwatt per centimeter square). Average whole body specific absorption rate (SAR) was calculated 0.1790 W/kg. After the exposure period the animals were sacrificed at different autopsy intervals viz., 1-30 days and the brain was excised to study various biochemical parameters. The animals were also tested in a radial arm maze to assess the learning and memory performance. Chronic exposure of these electromagnetic field radiations resulted in significant decrease in antioxidant enzyme activity of SOD (superoxide dismutase) and GSH (glutathione) with increased catalase activity (CAT) at all post treatment follow up intervals. Lipid peroxidation significantly increased at all the autopsy intervals. The protein content of the brain depleted upto 7th day after microwave exposure Radial arm maze test revealed that 10 GHz leads to significant depletion in spatial learning and memory performance in Swiss albino mice. Free radicals causing oxidative stress may be responsible for altered biochemical levels of the brain and impairment of learning and working memory of Swiss albino mice after microwave exposure.

Aflatoxins are carcinogenic secondary metabolites produced by the fungi Aspergillus flavus and Aspergillus parasiticus. Previous studies found that repeated serial mycelial transfer or treatment of A. parasiticus with 5-azacytidine produced colonies with a fluffy phenotype and inability to produce aflatoxins. To understand how these treatments affect expression of genes involved in aflatoxin production and development, we carried out expressed sequence tag (EST)-based microarray assays to identify genes in treated clones that are differentially expressed compared to the wild-type. Expression of 183 genes was significantly dysregulated. Of these, 38 had at least two-fold or lower expression compared to the untreated control and only two had two-fold or higher expression. The most frequent change was downregulation of genes predicted to encode membrane-bound proteins. Based on this result we hypothesize that the treatments cause changes in the structure of cellular and organelle membranes that prevent normal development and aflatoxin biosynthesis.

Trans-2-hexenal, a volatile aldehyde, is produced by soybean (Glycine max [L.] Merr) and other plants via the lipoxygenase pathway. In vitro tests showed it significantly (P < 0.001) reduced Aspergillus flavus germinating conidial viability at 10 μM, with approximately 95% viability reduction observed at 20 μM. The viability of nongerminated conidia was not reduced. To test the effectiveness of this volatile to prevent fungal growth in stored corn, trans-2-hexenal was pumped intermittently into glass jars containing corn. Experiments were performed to determine the ability of 2 different pump cycle time-courses to prevent A. flavus growth on sterile corn (23% moisture). Intermittently (30-min pumping period) over 7 d, this volatile was pumped through 350 g of corn kernels inoculated with 1 mL of 3 × 104 conidia of A. flavus. Controls consisted of (1) sterile corn, (2) corn inoculated with A. flavus with no pumped air, and (3) corn inoculated with A. flavus with intermittently pumped air. Aflatoxin B1 (AFB1), viability counts, and aldehyde concentration in the headspace were performed in each experiment. To determine whether an increased time period between volatile pumping would prevent A. flavus growth, a 2nd series of experiments were performed that were similar to the 1st series except that trans-2-hexenal (only) was pumped for a 30-min period every 12 h. Experiments were performed 3 times for each time course. Both experiments showed that intermittent pumping of volatile trans-2-hexenal significantly (P < 0.001) prevented A. flavus growth and aflatoxin B1 production over a 7-d period. Practical Application: Results from this study indicate that intermittent pumping of volatile trans-2-hexenal could be used to protect stored corn from A. flavus growth and aflatoxin contamination.

To better understand the effect of temperature on mycotoxin biosynthesis, RNA-Seq technology was used to profile the Aspergillus flavus transcriptome under different temperature conditions. This approach allowed us to quantify transcript abundance for over 80% of fungal genes including 1153 genes that were differentially expressed at 30 and 37 °C. Eleven of the 55 secondary metabolite clusters were upregulated at the lower temperature, including aflatoxin biosynthesis genes, which were among the most highly upexpressed genes. On average, transcript abundance for the 30 aflatoxin biosynthesis genes was 3300 times greater at 30 °C as compared with 37 °C. The results are consistent with the view that high temperature negatively affects aflatoxin production by turning down transcription of the two key transcriptional regulators, aflR and aflS. Subtle changes in the expression levels of aflS to aflR appear to control transcription activation of the aflatoxin cluster.

Wheat ( Triticum spp.) histones H1, H2, H3, and H4 were extracted, and H1 was further purified. The effect of these histones on specific fungi that may or may not be pathogenic to wheat was determined. These fungi included Aspergillus flavus , Aspergillus fumigatus , Aspergillus niger , Fusarium oxysporum , Fusarium verticillioides , Fusarium solani , Fusarium graminearum , Penicillium digitatum , Penicillium italicum , and Greeneria uvicola . Non-germinated and germinating conidia of these fungi were bioassayed separately. The non-germinated and germinating conidia of all Fusarium species were highly susceptible to the mixture (H1-H4) as well as pure H1, with viability losses of 99-100% found to be significant (p < 0.001) at ≤10 μM or less for the histone mixture and pure H1. F. graminearum was the most sensitive to histone activity. The histones were inactive against all of the non-germinated Penicillium spp. conidia. However, they significantly reduced the viability of the germinating conidia of the Penicillium spp. conidia, with 95% loss at 2.5 μM. Non-germinated and germinating conidia viability of the Aspergillus spp. and G. uvicola were unaffected when exposed to histones up to 10 μM. Results indicate that Fusarium spp. pathogenic to wheat are susceptible to wheat histones, indicating that these proteins may be a resistance mechanism in wheat against fungal infection.

α-Bisabolol, a natural plant sesquiterpene alcohol, and dragosantol, a racemic mixture of synthetic bisabolol, were studied for their fungicidal properties separately against the nongerminated and germinating conidia of several species of Aspergillus and Fusarium which represent problems in agriculture and medicine. Bactericidal assays against Pseudomonas aeruginosa and Staphylococcus aureus were also performed. Compared to the controls, both compounds displayed statistically significant (p < 0.001) lethality for the tested microorganisms. α-Bisabolol and dragosantol produced nearly 98% viability loss against the germinating conidia of A. flavus, A. fumigatus, A. niger, A. terreus, F. oxysporum, F. solani, and F. verticillioides (formerly F. moniliforme) at, or below, 10 μM. The viability of nongerminated F. oxysporum conidia was significantly reduced. Both compounds showed very rapid and significant reduction of P. aeruginosa viability at 7.5 μM. Staphylococcus aureus was more resistant to these...

Three soybean genotypes Kalitur, Hara soya and NRC37 with black, green and yellow seed coat color, respectively, were gamma irradiated at 0.5, 2.0 and 5.0 kGy and tested for antinutritional and nutritional factors. Gamma irradiation at all doses reduced the level of lipoxygenase isomers, trypsin inhibitor (TI) and ascorbic acid in all the 3 soybean genotypes as compared to the unirradiated control. However, irradiation dose of 5.0 kGy increased the sucrose content of the soybean genotypes. No significant change was observed in oil, protein fatty acids and total tocopherol content of the 3 genotypes at any irradiation dose. It is suggested that inhibition of lipoxygenase, reduction in TI and ascorbic acid may be due to the breakage or oxidation of protein structure by the gamma irradiation. Similarly, gamma irradiation at higher doses may break glycosidic linkages in oligosaccharides to produce more sucrose and decrease the content of flatulence causing oligosaccharides.

Toxigenic fungi invade crops prior to harvest as well as during storage and produce harmful, even carcinogenic toxins such as aflatoxins. Since consumers demand safe commodities, and due to enhanced public awareness of the dangers of many synthetic fungicides, the importance of investigating alternative, natural products to control these toxigenic fungi is clear. This study investigated the effect of aqueous extracts of Agave americana on growth, conidia and aflatoxin production. Aspergillus parasiticus strains SRRC 148, SRRC 143 (Su-1), and A. parasiticus SRRC 162, a mutant (nor-) that accumulates norsolorinic acid (NOR, an orange-coloured intermediate of the aflatoxin pathway), were first inoculated into Adye and Mateles liquid medium, then plant extracts were added, and incubated at 28 °C for 7 days. Aflatoxin and norsolorinic acid were assayed by HPLC and spectrophotometry, respectively. While the extract of A. americana stimulated growth of the studied fungi, conidiogenesis, norsolorinic acid accumulation (in the nor- mutant), and aflatoxin production were significantly affected. The reduction was produced by the extracts at concentrations higher than 5-10 mg/ml, where all types of total aflatoxin analysed (aflatoxins B1, B2, G1 and G2) were reduced from 64% to >99% in the whole culture, and a reduction of 75% of norsolorinic acid. The results of the present work indicate that extracts of A. americana may be promising safe alternatives to harmful fungicides for controlling aflatoxin contamination.

Biosynthesis of mycotoxins involves transcriptional co-regulation of sets of clustered genes. We hypothesise that specific control of transcription of genes in these clusters by LaeA, a global regulator of secondary metabolite production and development in many filamentous fungi, results from its interaction with a Cys6Zn2 DNA-binding protein unique to the gene cluster.

One strategy to reduce aflatoxin contamination of maize and cottonseed is to introduce spores of non-aflatoxigenic strains as competitors. Using isogenic mutants we show that, upon 5 or 20 min exposure to 302 nm (UVB) light, the viability of conidia of Aspergillus flavus and Aspergillus parasiticus mutants lacking the ability to accumulate any aflatoxin precursor metabolite is reduced five-fold compared to that of aflatoxin-producing strains or pigmented mutants that accumulate aflatoxin precursors. This result suggests that the long-term viability of introduced non-aflatoxigenic competitor strains may be lower than that of natural aflatoxin-producing isolates when exposed to sunlight.