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[Display omitted] • 23 species of section Flavi, 2 of Ochraceorosei, and 4 of Nidulantes are aflatoxigenic. • aflR , the main aflatoxin regulator gene doesn't confirm Aspergilli as aflatoxigenic. • Evolutional variation, telomere deletion, and missense mutation in gene cluster makes Aspergilli non-aflatoxigenic. • Aspergilli in each section show groupism without intrusion from other group species. • Species of section Ochraceorosei have better genomic profile for aflatoxin synthesis. Aspergillus species produce polyketides, which form the basis of aflatoxins, some of the most significant mycotoxins in agriculture. Aflatoxins contaminate cereals, oilseeds, and nuts, both in the field and during storage. Of the 13 naturally occurring aflatoxins, the most potent are aflatoxins B 1 , B 2 , G 1 , and G 2. The primary aflatoxigenic species are A. flavus, A. parasiticus , and A. nomius, while A. arachidicola , A. minisclerotigenes , and A. saccharicola also documented. These aflatoxin producers belong to three sections- 'Flavi', 'Ochraceorosei', and 'Nidulantes.' Aspergillus flavus , within section Flavi, shows morphological diversity, classified into Group I (S- and L- strains) and Group II (S- strains), with S-strains producing higher levels of aflatoxins. Aflatoxin biosynthesis is primarily regulated by the aflR gene, though other genes like aflS, aflP, aflQ, aflC, and aflM are also associated. However, presence of the aflR gene does not guarantee aflatoxin production across species. Sterigmatocystin serves as a precursor molecule within the pathway leading to aflatoxin production. Phylogenetic assessment, using ITS, BenA , CaM , and RBP2 gene sequences, reveals distinct clusters within Aspergillus sections and highlights the co-evolution of aflatoxigenic and non-aflatoxigenic species. Aspergillus ochraceoroseus and A. rambellii diverged out of aflatoxin-producing species earlier in evolutionary history, before splitting from a shared ancestor with A. fumigatus , which neither produces aflatoxins nor sterigmatocystin. Non-aflatoxigenic species like A. oryzae may evolve from aflatoxigenic species like A. flavus due to variations in evolutionary rates, telomere deletions, and mutations in aflatoxin biosynthesis genes. Comparative genomic analysis of AF, AF/ST and ST gene cluster shows that A. flavus has a larger aflatoxin gene cluster, while A. ochraceoroseus lacks the genes aflP and aflQ. Additionally, A. ochraceoroseus and A. rambellii possess a smaller genome, suggesting that genetic drift and deletions have refined their genomes for more efficient aflatoxin production. [ABSTRACT FROM AUTHOR]

Organisms are repeatedly exposed to fluctuating environmental and nutritional conditions. Transcriptional memory has been shown to be a mechanism to cope with these fluctuations because it increases the speed and the magnitude of the cellular response to a certain re-occurring condition and therefore optimizes adaptation and fitness in a given environment. We found that genes coding for sterigmatocystin (ST) production in Aspergillus nidulans are activated stronger when cells are repeatedly exposed to nutrient starvation, compared to cells that experience this condition for the first time. We studied possible underlying mechanisms and found that persistence of the transcription factor AflR, which can undergo activation-inactivation cycles, accounts for a large part of the memory. In addition, a chromatin-based mechanism through histone H3 lysine 4 dimethylation (H3K4me2) and extracellular metabolites produced during the first activation phase contribute to the memory process. Genome-wide transcriptome and chromatin analyses showed that only a few genes within the ST and other starvation-induced biosynthetic gene clusters gain the H3K4me2 mark during the 1st activation, but the majority of those which receive the mark also maintain it during the subsequent repression and re-activation phase. Combined with previous findings on chromatin-level regulation of biosynthetic gene clusters (BGCs) our recent data suggest that the H3K4me2 mark may contribute to the correct 3D organization of BGCs and that this is a prerequisite for activation and transcriptional memory. [ABSTRACT FROM AUTHOR]

ABSTRACT Aspergillus flavus aflR, a gene encoding a Zn(II)2Cys6 DNA-binding domain, is an important transcriptional regulator of the aflatoxin biosynthesis gene cluster. Our previous results of Gene ontology (GO) analysis for the binding sites of AflR in A. flavus suggest that AflR may play an integrative regulatory role. In this study the ΔaflR and overexpression (OE) strains based on the well-established double-crossover recombinational technique were constructed to investigate the integrative function of the aflR gene in A. flavus. The disruption of aflR severely affected the aflatoxin biosynthetic pathway, resulting in a significant decrease in aflatoxin production. The aflatoxin B1 (AFB1) of the ΔaflR strain was 180 ng/mL and aflatoxin B2 (AFB2) was 2.95 ng/mL on YES medium for 5 days, which was 1/1,000 of that produced by the wild-type strain (WT). In addition, the ΔaflR strain produced relatively sparse conidia and a very small number of sclerotia. On the seventh day, the sclerotia yield on each plate of the WT and OE strains exceeded 1,000, while the sclerotial formation of the ΔaflR strain was not detected until 14 days. However, the biosynthesis of cyclopiazonic acid (CPA) was not affected by aflR gene disruption. Transcriptomic analysis of the ΔaflR strain grown on potato dextrose agar (PDA) plates at 0 h, 24 h, and 72 h showed that expression of clustering genes involved in the biosynthesis of aflatoxin was significantly downregulated. Meanwhile, the ΔaflR strain compared with the WT strain showed significant expression differences in genes involved in spore germination, sclerotial development, and carbohydrate metabolism compared to the WT. The results demonstrated that the A. flavus aflR gene also played a positive role in the fungal growth and development in addition to aflatoxin biosynthesis. IMPORTANCE Past studies of the A. flavus aflR gene and its orthologues in related Aspergillus species were solely focused on their roles in secondary metabolism. In this study, we used the ΔaflR and OE strains to demonstrate the role of aflR in growth and development of A. flavus. For the first time, we confirmed that the ΔaflR strain also was defective in production of conidia and sclerotia, asexual propagules of A. flavus. Our transcriptomic analysis further showed that genes involved in spore germination, sclerotial development, aflatoxin biosynssssthesis, and carbohydrate metabolism exhibited significant differences in the ΔaflR strain compared with the WT strain. Our study indicates that AflR not only plays an important role in regulating aflatoxin synthesis but also in playing a positive role in the conidial formation and sclerotial development in A. flavus. This study reveals the critical and positive role of the aflR gene in fungal growth and development, and provides a theoretical basis for the genetic studies of other aspergilli.

Aflatoxins (AFs), potent foodborne carcinogens produced by Aspergillus fungi, pose significant health risks worldwide and present challenges to food safety and productivity in the food chain. Novel strategies for disrupting AF production, cultivating resilient crops, and detecting contaminated food are urgently needed. Understanding the regulatory mechanisms of AF production is pivotal for targeted interventions to mitigate toxin accumulation in food and feed. The gene cluster responsible for AF biosynthesis encodes biosynthetic enzymes and pathway-specific regulators, notably AflR and AflS. While AflR, a DNA-binding protein, activates gene transcription within the cluster, AflS enhances AF production through mechanisms that are not fully understood. In this study, we developed protocols to purify recombinant AflR and AflS proteins and utilized multiple assays to characterize their interactions with DNA. Our biophysical analysis indicated that AflR and AflS form a complex. AflS exhibited no DNA-binding capability on its own but unexpectedly reduced the DNA-binding affinity of AflR. Additionally, we found that AflR achieves its binding specificity through a mechanism in which either two copies of AflR or its complex with AflS bind to target sites on DNA in a highly cooperative manner. The estimated values of the interaction parameters of AflR, AflS and DNA target sites constitute a fundamental framework against which the function and mechanisms of other AF biosynthesis regulators can be compared., (© 2024 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

The aim was to decipher the temporal impact of key interacting climate change (CC) abiotic factors of temperature (30 vs 37 °C), water activity (a w ; 0.985 vs 0.930) and CO 2 exposure (400 vs 1000 ppm) on (a) growth of Aspergillus flavus and effects on (b) gene expression of a structural (aflD) and key regulatory gene (aflR) involved in aflatoxin B 1 (AFB 1) biosynthesis and (c) AFB 1 production on a yeast extract sucrose medium over a period of 10 days. A. flavus grew and produced AFB 1 very early with toxin detected after only 48 h. Both growth and toxin production were significantly impacted by the interacting abiotic factors. The relative expression of the aflD gene was significantly influenced by temperature; aflR gene expression was mainly modulated by time. However, no clear relationship was observed for both genes with AFB 1 production over the experimental time frame. The optimum temperature for AFB 1 production was 30 °C. Maximum AFB 1 production occurred between days 4–8. Exposure to higher CO 2 conditions simulating forecasted CC conditions resulted in the amount of AFB 1 produced in elevated temperature (37 °C) being higher than with the optimum temperature (30 °C) showing a potential for increased risk for human/animal health due to higher accumulation of this toxin. [ABSTRACT FROM AUTHOR]

We report here the AflR binding motif of Aspergillus flavus for the first time with the aid of ChIP-seq analysis. Of the 540 peak sequences associated with AflR binding events, 66.8% were located within 2 kb upstream (promoter region) of translational start sites. The identified 18-bp binding motif was a perfect palindromic sequence, 5′-CSSGGGWTCGAWCCCSSG’3′ with S representing G or C and W representing A or T. On closer examination, we hypothesized that the 18-bp motif sequence identified contained two identical parts (here called motif A and motif B). Motif A was in positions 8–18 on the upper strand, while motif B was in positions 11-1 on the bottom strand. The inferred length and sequence of the putative motif identified in A. flavus were similar to previous findings in A. parasiticus and A. nidulans. Gene ontology analysis indicated that AflR bound to other genes outside the aflatoxin biosynthetic gene cluster. [ABSTRACT FROM AUTHOR]

Aflatoxin B 1 (AFB 1) is one of the most hazardous mycotoxins for humans and livestock that mainly produced by members of the genus Aspergillus in a variety of food commodities. In this study, the effect of S. rosmarinus , T. fruticulosum , and T. caucasicum essential oils (EOs) was studied on fungal growth, AFB 1 production and aflR gene expression in toxigenic A. flavus IPI 247. The AFB 1 producer A. flavus strain was cultured in YES medium in presence of various two-fold concentrations of the plant EOs (62.5–500 μg/mL) for 4 days at 28 °C. EO composition of plants was analyzed by Gas Chromatography/Mass Spectrometry (GC/MS). The amount of fungal growth, ergosterol content of fungal mycelia and AFB 1 content of EO-treated and non-treated controls were measured. The expression of aflR gene was evaluated using Real-time PCR in the fungus exposed to minimum inhibitory concentration (MIC 50) of EOs. The main constituents of the oils analyzed by GC/MS analysis were elemicin (33.80 %) and 2,3-dihydro farnesol (33.19 %) in T. caucasicum , 1,8-cineole (17.87 %), trans-caryophyllene (11.14 %), α and ẞ-pinene (10.92 and 8.83 %) in S. rosmarinus , and camphor (17.65 %), bornyl acetate (15.08 %), borneol (12.48 %) and camphene (11.72 %) in T. fruticulosum. The results showed that plant EOs at the concentration of 500 μg/mL suppressed significantly the fungal growth by 35.24–71.70 %, while mycelial ergosterol content and AFB 1 production were inhibited meaningfully by 36.20–65.51 % and 20.61–89.16 %. T. caucasicum was the most effective plant, while T. fruticulosum showed the lowest effectiveness on fungal growth and AFB 1 production. The expression of aflR in T. caucasicum and S. rosmarinus -treated fungus was significantly down-regulated by 2.85 and 2.12 folds, respectively, while it did not change in T. fruticulosum -treated A. flavus compared to non-treated controls. Our findings on the inhibitory activity of T. caucasicum and S. rosmarinus EOs toward A. flavus growth and AFB 1 production could promise these plants as good candidates to control fungal contamination of agricultural crops and food commodities and subsequent contamination by AFB 1. Down-regulation of aflR as the key regulatory gene in AF biosynthesis pathway warrants the use of these plants in AF control programs. Further studies to evaluate the inhibitory activity of studied plants EOs in food model systems are recommended. [Display omitted] • EOs of T. caucasicum , S. rosmarinus and T. fruticulosum analysed by GC/MS inhibited A. flavus growth and AFB 1 production. • Ergosterol content of fungal mycelia was diminished significantly by the plants EOs. • Fungal growth, AFB 1 synthesis and ergosterol production were inhibited by plant Eos time-dependently. • aflR gene expression was strongly suppressed by T. caucasicum and S. rosmarinus. • These bioactive plants would be promising to control fungal growth and subsequent AFB 1 contamination of food and feed. [ABSTRACT FROM AUTHOR]

In this paper we aim to deliver a key message related to the safeguarding of the Romanian National Phonogram Archive (AFLR). The data gathered within the Archive (the richest, most inclusive and diversified collection of dialectal texts and ethno-linguistic recordings in Romania) are of immeasurable documentary value. Through the digitization and preservation of AFLR we can gain access to both individual and collective memories, aiding to a better understanding of our cultural heritage on the one hand, and, on the other hand, restoring missing or forgotten pieces of Europe's oral history. [ABSTRACT FROM AUTHOR]

Pistachio nuts are an important economic tree nut crop which is used directly or processed for many food-related activities. They can become colonized by mycotoxigenic spoilage fungi, especially Aspergillus flavus, mainly resulting in contamination with aflatoxins (AFs), especially aflatoxin B1 (AFB1). The prevailing climate in which these crops are grown changes as temperature and atmospheric CO2 levels increase, and episodes of extreme wet/dry cycles occur due to human industrial activity. The objectives of this study were to evaluate the effect of interacting Climate Change (CC)-related abiotic factors of temperature (35 vs. 37 °C), CO2 (400 vs. 1000 ppm), and water stress (0.98–0.93 water activity, aw) on (a) growth (b) aflD and aflR biosynthetic gene expression and (c) AFB1 production by two strains A. flavus (AB3, AB10) in vitro on milled pistachio-based media and when colonizing layers of shelled raw pistachio nuts. The A. flavus strains were resilient in terms of growth on pistachio-based media and the colonisation of pistachio nuts with no significant difference when exposed to the interacting three-way climate-related abiotic factors. However, in vitro studies showed that AFB1 production was significantly stimulated (p < 0.05), especially when exposed to 1000 ppm CO2 at 0.98–0.95 aw and 35 °C, and sometimes in the 37 °C treatment group at 0.98 aw. The relative expression of the structural aflD gene involved in AFB1 biosynthesis was decreased or only slightly increased, relative to the control conditions at elevated CO, regardless of the aw level examined. For the regulatory aflR gene expression, there was a significant (p < 0.05) increase in 1000 ppm CO2 and 37 °C for both strains, especially at 0.95 aw. The in situ colonization of pistachio nuts resulted in a significant (p < 0.05) stimulation of AFB1 production at 35 °C and 1000 ppm CO2 for both strains, especially at 0.98 aw. At 37 °C, AFB1 production was either decreased, in strain AB3, or remained similar, as in strain AB10, when exposed to 1000 ppm CO2. This suggests that CC factors may have a differential effect, depending on the interacting conditions of temperature, exposure to CO2 and the level of water stress on AFB1 production.

Abstract The regulatory gene (aflR) encodes AflR, a positive regulator of transcriptional pathway that activates aflatoxin biosynthesis. It has been demonstrated in our laboratory that L-Asp-L-Asn (DN) extracted from Bacillus megaterium inhibited the growth of Aspergillus flavus. We fused gene encoding DN with the gene encoding specific dinuclear zinc finger cluster protein of AflR, then fusion protein competed with the AflS-AflR complex for the AflR binding site and significantly improved anti- A. flavus activity (growth of A. flavus and biosynthesis of aflatoxin B 1) of DN. The fusion gene dn-aflR was cloned into pET32a and recombinant plasmid was introduced into Escherichia coli BL21. The highest expression was observed after 10 h induction and fusion protein was purified by affinity chromatography column. Compared with DN, the novel fusion protein DN-AflR significantly inhibited the growth of A. flavus and biosynthesis of aflatoxin B 1 (P < 0.05). This study promoted the use of competitive inhibition of fusion proteins to reduce the expression of regulatory genes in the biosynthetic pathway of aflatoxin. Moreover, it provided more supports for deep research and industrialization of such novel anti- A. flavus bio-inhibitors and biological control of microbial contamination. Highlights • Plasmid dn-aflR-pET32a containing the genes encoding DN and zinc finger cluster protein has been designed and constructed • The novel fusion protein DN-AflR significantly inhibited the growth of A. flavus and biosynthesis of aflatoxin B 1 • This research showed fusion expression is a promising method to improve inhibitory activity of bio-inhibitors for A. flavus. [ABSTRACT FROM AUTHOR]

Aspergillus nidulans produces sterigmatocystin (ST), a precursor of a carcinogenic secondary metabolite aflatoxin (AF), during its developmental process. ST biosynthesis has been shown to be affected by various regulatory factors. In this study, we investigated the involvement of O-mannosyltransferases (PmtA, PmtB, PmtC), in ST production and morphological development. Deletion of pmtA (ΔpmtA), pmtB (ΔpmtB) or pmtC (ΔpmtC) caused no spore production and a significant decline of vegetative growth. A tremendous decline of ST level was observed in all Δpmt mutants at the third day after inoculation. By extending the growth period, ST production of ΔpmtA and ΔpmtB increased to the wild-type level 7 days after inoculation. On the other hand, ST was not detected from 7- or 14-day cultures in ΔpmtC. Expression levels of aflR gene, an essential regulator of the ST biosynthesis pathway, were also down-regulated in the Δpmt strains. By introducing the aflR overexpression cassette, ST production in the ΔpmtA and ΔpmtB significantly increased to levels comparable to the wild type. However, the presence of the aflR overexpression cassette could not improve ST production in the ΔpmtC mutant. These data suggest that the PMT family is a new endogenous factor that is required for ST biosynthesis in A. nidulans. These findings provide better understanding of the regulatory mechanisms of AF/ST biosynthesis, which can ultimately contribute to our ability to control aflatoxin contamination. [ABSTRACT FROM AUTHOR]

Maize is prone to infection by Aspergillus flavus , which can contaminate the product with aflatoxins. The objective of this study was to examine the impact that interactions between water activity (a w ) and temperature may have on growth, the expression of a biosynthetic regulatory gene ( aflR ) and aflatoxin B 1 (AFB 1 ) production by a strain of A. flavus on a maize-based medium. Results showed that there were some differences between lag phases and growth rates of A. flavus . The optimum growth rate for A. flavus was at 30 °C and 0.99 a w . No growth occurred at 0.90 a w and 20 °C. Both temperature and a w had an influence on the relative aflR gene expression and AFB 1 production by A. flavus ; however, the results for AFB 1 production were not consistent with the effects on gene expression or growth. These findings provide data that are useful to better understand the conditions which represent higher risks from AFB 1 production. However, the aflR expression was not a good indicator of AFB 1 production alone. Thus, further molecular studies of other AF-related genes should be done. These results are discussed in the context of harvesting and storage of maize and the prevailing environmental conditions to minimise AFB 1 contamination. [ABSTRACT FROM AUTHOR]

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.

"nBackground: Aspergillus flavus is the second most important Aspergillus species causing human infections particu­larly fun­gal sinusitis. Since little is known about aflatoxin producing ability of clinical isolates, this study was under­taken to de­tect the aflatoxigenic isolates amongst these isolates."nMethods: A total of 23 isolates of A. spp. which were recovered from patients proved to have fungal sinusitis by morpho­logi­cal and histological methods and also 5 additional aflatoxigenic and non-aflatoxigenic reference of A. fla­vus group strains were studied. The isolates were identified morphologically using Czapek Yeast Agar and A. flavus and parasiticus Agar (AFPA). Aflatoxin producing ability of the isolates was confirmed by Thin Layer Chromatogra­phy. Existing of aflR gene the regulatory gene in aflatoxin biosynthesis, were studied in all isolates by PCR method."nResults: All twenty three Aspergillus isolates confirmed as A. flavus group by their macroscopic and microscopic fea­tures. One clinical isolate confirmed as A. oryzae by mycological methods. A. oryzae as well as A. flavus JCM2061 and NCPF2008 and 3 clinical isolates were not able to produce orange pigment on AFPA. From total of 23 iso­lates 4 (17.4%) con­firmed to be aflatoxigenic by TLC method. A banding pattern which matched to aflR primers was amplified with approxi­mate size of 800 bp in all 23 clinical A. flavus isolates. A larger banding pattern 1050 bp was revealed in clinical iso­late; strain no.20 as well."nConclusion: Some clinical sinus isolates are able to produce aflatoxin and all of studied isolates including; A. oryzae, A. parasiti­cus and A. sojae were able to amplify aflR gene under our laboratory conditions.

The objective of this study was to carry out a transcription analysis of eight genes belonging to the aflatoxin (AF) and cyclopiazonic acid (CPA) biosynthesis pathway, and to detect aflatoxin B 1 (AFB 1 ) and CPA production in Aspergillus flavus strains isolated from Brazil nuts. Additionally, these genes were correlated with the different mycotoxigenic profiles of the same strains. Four previously identified A. flavus strains (ICB-01, ICB-151, ICB-161, and ICB-165) were grown on Brazil nut agar at 25 °C for 10 days. Mycotoxins were separated by high-performance liquid chromatography. Transcriptional analysis was performed by real-time RT-PCR using specific primers designed based on the conserved regions of two regulatory genes ( aflR and aflS ), three structural genes of the AFB 1 biosynthesis pathway ( aflH , aflJ and aflP ), and three structural genes of the CPA biosynthesis pathway ( maoA , dmaT and pks-nrps ). The expression of most genes in the A. flavus isolates varied according to the mycotoxin profile of each strain. The most expressed genes in the aflatoxigenic strain ICB-151 were aflJ (77.11%) and aflH (32.75%), while the CPA-producing strain ICB-161 mainly expressed dmaT (100%), maoA (63.72%), aflS (43.52%), and aflR (42.63%). The ICB-01 isolate was a producer of AFB 1 and CPA and the most expressed genes were aflS ( 47.79%), dmaT (42.77%), aflP (39.5%), and aflR (38.02%). ICB-198 did not produce any mycotoxin and exhibited lower expression of almost all genes analyzed. Furthermore, the ratio of aflS / aflR expression was correlated with the biosynthesis of AF and CPA in A. flavus strains producing exclusively AF or CPA or producing both AF and CPA. The ratio of aflS / aflR expression therefore seems to be related to the production of mycotoxins in Brazil nuts. Our results provide important data for the development of innovative and more cost-effective strategies to reduce and prevent AFB and CPA contamination in Brazil nuts. [ABSTRACT FROM AUTHOR]

Variation in grazing intensity determines the way in which mycelial growth and physiology is influenced by invertebrate fungivores. Recently, low-intensity grazing by facultative mesofaunal fungivores, small dipteran larvae and collembola, was found to enhance the activity of fungal defensive secondary metabolism. Here, we ask whether higher intensity grazing by macrofaunal fungivores induces a different fungal response. To test this, we quantified changes in the expression of two anti-fungivore chemical defence marker genes, laeA and aflR , in the ascomycete Aspergillus nidulans exposed to grazing pressure exerted by individuals of the isopod Oniscus asellus . Compared to no and low-intensity grazing pressure, higher intensity grazing significantly reduced the transcript levels of these genes. This finding suggests a negative relationship between grazing pressure intensity and the capacity of a saprotrophic fungus to resist fungivory. [ABSTRACT FROM AUTHOR]

An aspect of mycotoxin biosynthesis that remains unclear is its relationship with the cellular management of reactive oxygen species (ROS). Here we conduct a comparative study of the total ROS production in the wild-type strain (SU-1) of the plant pathogen and aflatoxin producer, Aspergillus parasiticus, and its mutant strain, AFS10, in which the aflatoxin biosynthesis pathway is blocked by disruption of its pathway regulator, aflR. We show that SU-1 demonstrates a significantly faster decrease in total ROS than AFS10 between 24 h to 48 h, a time window within which aflatoxin synthesis is activated and reaches peak levels in SU-1. The impact of aflatoxin synthesis in alleviation of ROS correlated well with the transcriptional activation of five superoxide dismutases (SOD), a group of enzymes that protect cells from elevated levels of a class of ROS, the superoxide radicals (O2−). Finally, we show that aflatoxin supplementation to AFS10 growth medium results in a significant reduction of total ROS only in 24 h cultures, without resulting in significant changes in SOD gene expression. Our findings show that the activation of aflatoxin biosynthesis in A. parasiticus alleviates ROS generation, which in turn, can be both aflR dependent and aflatoxin dependent.

The objectives of this study were to examine the efficacy of four bacterial antagonists against Aspergillus flavus using 50:50 ratio of bacterial cells/conidia for the control of aflatoxin B 1 (AFB 1 ) production on two different nutritional matrices, nutrient and maize-based media at different water availabilities (0.98, 0.94 water activity (a w ) on nutrient medium; 0.995, 0.98 a w on maize meal agar medium) at 35 °C. The indicators of efficacy used were the relative expression of one structural and regulatory gene in the biosynthetic pathway ( aflD and aflR respectively) and the production of AFB 1 . These studies showed that some of the bacterial species could significantly inhibit the relative expression of the aflD and aflR genes at both 0.98 and 0.94 a w on nutrient agar. On maize-based media some of the bacterial antagonists reduced the activity of both genes at 0.94 a w and some at 0.995 a w . However, the results for AFB 1 production were not consistent with the effects on gene expression. Some bacterial species stimulated AFB 1 production on both nutrient and maize-based media regardless of a w . However, some bacterial treatments did inhibit AFB 1 production significantly when compared to the control. Overall, this study suggests that temporal studies are required on the biosynthetic genes under different environmental and nutritional conditions to evaluate the potential of antagonists to control AFB 1 . [ABSTRACT FROM AUTHOR]

Key message: We report success of host-induced gene silencing in downregulation of aflatoxin biosynthesis in Aspergillus flavus infecting maize transformed with a hairpin construct targeting transcription factor aflR. Abstract: Infestation of crops by aflatoxin-producing fungi results in economic losses as well as negative human and animal health effects. Currently, the control strategies against aflatoxin accumulation are not effective to the small holder farming systems in Africa and this has led to widespread aflatoxin exposure especially in rural populations of sub-Saharan Africa that rely on maize as a staple food crop. A recent strategy called host-induced gene silencing holds great potential for developing aflatoxin-resistant plant germplasm for the African context where farmers are unable to make further investments other than access to the germplasm. We transformed maize with a hairpin construct targeting the aflatoxin biosynthesis transcription factor aflR. The developed transgenic maize were challenged with an aflatoxigenic Aspergillus flavus strain from Eastern Kenya, a region endemic to aflatoxin outbreaks. Our results indicated that aflR was downregulated in A. flavus colonizing transgenic maize. Further, maize kernels from transgenic plants accumulated significantly lower levels of aflatoxins (14-fold) than those from wild type plants. Interestingly, we observed that our silencing cassette caused stunting and reduced kernel placement in the transgenic maize. This could have been due to 'off-target' silencing of unintended genes in transformed plants by aflR siRNAs. Overall, this work indicates that host-induced gene silencing has potential in developing aflatoxin-resistant germplasm. [ABSTRACT FROM AUTHOR]

Three types of media and a multiplex PCR procedure with a set of four primers were used to differentiate between aflatoxinogenic and non-aflatoxinogenic strains of Aspergillus flavus and Aspergillus parasiticus. Four sets of primers were the aflR, nor-1, ver-1, and omt-A genes of the aflatoxin biosynthetic pathway. Multiplex PCR showed that the four aflatoxinogenic strains gave a quadruplet pattern, indicating the presence of all the genes involved in the aflatoxin biosynthetic pathway which encode for the products. Non-aflatoxinogenic strains gave varying results with two, three, or four banding patterns. A banding pattern in seven non-aflatoxinogenic strains resulted in non-differentiation between these and aflatoxinogenic strains. [ABSTRACT FROM AUTHOR]

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 Δ?aflJ 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. [ABSTRACT FROM AUTHOR]

Abstract: Aspergillus parasiticus mutant strains resistant to DMIs were isolated in a high mutation frequency after UV-mutagenesis and selection on media containing flusilazole. Two different resistant phenotypes, R1 and R2, on the basis of their aflatoxigenic ability were identified. All R1 mutant strains produced aflatoxins at concentrations significantly higher (up to 3-fold) than the wild-type parent strain on yeast extract sucrose medium, whereas the majority of mutant strains (R2 phenotype) lost their aflatoxigenic ability. Real-time PCR analysis of the expression levels of the aflR gene, a pathway transcriptional regulatory gene in aflatoxin biosynthesis, showed that this gene was not expressed in R2 mutant strains tested. Study of fitness determining parameters showed that most flusilazole-resistant mutant strains had mycelial growth rate, sporulation and spore germination lower that the sensitive one. Cross-resistance studies with other fungicides showed that all R1 mutant strains were also resistant to the DMIs imazalil and tebuconazole, but retained their parental sensitivity to fungicides affecting other metabolic pathways and/or cellular processes. Contrary to the above, all R2 mutant strains exhibited a low to moderate multi-drug resistance to DMIs and to several other fungicide classes. Two different homologous genes, cyp51A and cyp51B, encoding C-14 alpha sterol demethylase (Cyp51) and an mdr gene encoding an ATP-binding cassette protein which may be involved in multidrug resistance were cloned and characterized. Sequence comparison of cyp51A gene revealed an amino acid substitution from glycine (GGG) to tryptophan (TGG) at position 54 (G54W) in two out of three of R1 mutant strains. Analysis of deduced amino acid sequence of cyp51B showed that no mutations were associated with DMI resistance. Study for the transcriptional levels of cyp51A showed that this gene was over-expressed in the third aflatoxigenic mutant strain. Neither amino acid substitutions nor an overexpression of the cyp51A gene were found in the R2 mutant strains tested. Real-time PCR analysis showed high levels (up to 25-fold higher) of the mdr transcript in all R2 mutant strains tested. This is the first report describing the existence of two cyp51 genes and a potential mdr gene coding for an ATP binding cassette protein in A. parasiticus. These results also indicate that multiple biochemical mechanisms, including target-site modification due to mutation at cyp51A gene, overexpression of cyp51A gene and the function of an ABC transporter protein, are responsible for DMI-resistance in A. parasiticus. Our findings suggest that A. parasiticus have the genetic and biochemical potential for the appearance of highly aflatoxigenic DMI-resistant isolates in the field. [Copyright &y& Elsevier]

Abstract: LaeA of Aspergillus nidulans is a putative methyltransferase and a component of the velvet complex; it is thought to mainly affect expression of genes required for the production of secondary metabolites. We found that although Aspergillus flavus CA14 laeA deletion mutants showed no aflatoxin production, expression of some of the early genes involved in aflatoxin formation, but not the later genes, could still be detected. The mutants grown in minimal medium supplemented with simple sugars and on some complex media exhibited altered conidial development. On potato dextrose agar (PDA) medium the deletion mutants showed reduced conidial chain elongation, increased production of conidiophores, and decreased colony hydrophobicity when compared to the parental strain. The loss of hydrophobicity and the other developmental changes in the laeA deletion mutants could affect the ability of the fungus to produce aflatoxins. [Copyright &y& Elsevier]

Abstract: Aspergillus oryzae, although closely related to Aspergillus flavus, does not produce aflatoxin (AF). A. oryzae RIB strains can be classified into three groups (group 1–3) based on the structure of the AF biosynthesis gene homolog cluster (AFHC). In group 1 strains, where AFHC is present, the expression level of the aflR gene is extremely low and there is no expression of the other four AF homologue genes (avnA, verB, omtA and vbs). We conducted a detailed structural comparison of AFLR ORF and AFLJ ORF from A. oryzae and A. flavus and identified several amino-acid substitutions. If these substitutions induce inactivation of AFLR and AFLJ, AF biosynthesis of A. oryzae will be doubly inhibited at the transcriptional and translational level. In this study, we transferred aflR and aflJ to A. oryzae RIB67, a group 2 strain where more than half of AFHC is missing. Under control of the pgkA promoter, aflR and aflJ was expressed and avnA, verB, omtA and vbs gene expression were monitored by RT-PCR. We prepared six types of forced-expression vectors, including aflR (from A. oryzae RIB40 or its three mutants) or aflJ (from A. oryzae RIB40 or A. flavus RIB4011). RT-PCR analysis showed that transformants containing aflJ from A. oryzae displayed no expression of AF biosynthetic homologue genes, whereas aflR substitutions had no such effect. These results strongly suggest that the amino-acid substitutions in AFLJ of A. oryzae induce inactivation at the protein level. [Copyright &y& Elsevier]

Abstract: The nuclear regulator LaeA has been shown to govern production of multiple secondary metabolites in Aspergillus nidulans and Aspergillus fumigatus. Herein we examine the role of this protein in Aspergillus flavus. Similarly as in other Aspergilli, LaeA had a major effect on A. flavus secondary metabolism where ΔlaeA and over-expression laeA (OE::laeA) strains yielded opposite phenotypes resulting in decreased (increased) secondary metabolite production. The two mutant strains also exhibited striking morphological phenotypes in the loss (increase) of sclerotial production in comparison to wildtype. Growth on seed was marked by decreased (increased) conidial and aflatoxin production of the respective mutants; this was accompanied by decreased lipase activity in ΔlaeA, an enzymatic process correlated with seed maceration. Transcriptional examination of the mutants showed LaeA negatively regulates expression of its recently identified nuclear partner VeA, another global regulator of A. flavus secondary metabolites and sclerotia. [Copyright &y& Elsevier]

A genetic linkage map of black gram, Vigna mungo (L.) Hepper, was constructed with 428 molecular markers using an F9 recombinant inbred population of 104 individuals. The population was derived from an inter-subspecific cross between a black gram cultivar, TU94-2, and a wild genotype, V. mungo var. silvestris. The linkage analysis at a LOD score of 5.0 distributed all 428 markers (254 AFLP, 47 SSR, 86 RAPD, and 41 ISSR) into 11 linkage groups. The map spanned a total distance of 865.1 cM with an average marker density of 2 cM. The largest linkage group spanned 115 cM and the smallest linkage group was of 44.9 cM. The number of markers per linkage group ranged from 11 to 86 and the average distance between markers varied from 1.1 to 5.6 cM. Comparison of the map with other published azuki bean and black gram maps showed high colinearity of markers, with some inversions. The current map is the most saturated map for black gram to date and will provide a useful tool for identification of QTLs and for marker-assisted selection of agronomically important characters in black gram. Une carte génétique du haricot mungo, Vigna mungo (L.) Hepper, a été construite à l’aide de 428 marqueurs moléculaires sur une population de 104 lignées recombinantes F9. La population est dérivée d’un croisement entre deux sous-espèces, le cultivar de haricot mungo TU94-2 et un génotype sauvage du V. mungo var. silvetris. L’analyse de liaison réalisée à un score LOD de 5,0 a permis de distribuer les 428 marqueurs (254 AFLP, 47 SSR, 86 RAPD et 41 ISSR) sur 11 groupes de liaison. La carte totalise 865,1 cM et présente un marqueur aux 2 cM en moyenne. Le plus grand groupe de liaison s’étend sur 115 cM et le plus petit compte 44,9 cM. Le nombre de marqueurs par groupe de liaison varie entre 11 et 86 tandis que la distance entre marqueurs varie entre 1,1 et 5,6 cM. La comparaison de cette carte avec celles publiées auparavant pour les haricots mungo et azuki a montré une importante colinéarité quant à l’ordre des marqueurs avec quelques inversions. La présente carte est la plus saturée à ce jour pour le haricot mungo et sera utile pour l’identification de QTL et la sélection assistée de marqueurs pour des caractères d’intérêt agronomique chez le haricot mungo. [ABSTRACT FROM AUTHOR]

To estimate the incidence contamination of fresh pistachio nuts by aflatoxigenic fungi in Iran, nut samples were collected from pistachio orchards in Kerman, Rafsanjan and Isfahan regions. Out of the 200 Aspergillus isolates obtained, 11 species were identified as A. alliaceous, A. candidus, A. flavus, A. niger, A. niveus, A. ochraceus, A. parasiticus, A. tamari, A. terreus, A. unguis and A. wentii. For detection of aflatoxin production ability of the isolates, three target genes, namely aflR, aflJ, and omtB, used in PCR amplification. In all the examined cases, the degenerate primer designed for amplification of omtB gene, named omtBII, was able to amplify an expected 611 bp fragment in aflatoxigenic isolates in this study and yielded the same result as those obtained from TLC analysis and fluorescence ability by application of methylated β-cyclodextrin in culture media. Using this procedure the significant incidence of aflatoxin-producing aspergilli was confirmed in pistachio nuts produced in different regions of Iran. The results indicated that PCR method described here, in combination with fluorescence assay, is a reliable and simple confirmatory test for monitoring pistachio nuts contaminated with aflatoxinogenic aspergilli. [ABSTRACT FROM AUTHOR]

The molecular regulation of aflatoxin biosynthesis is complex and influenced by several environmental conditions; one of these is temperature. Aflatoxins are produced optimally at 28-30 C, and production decreases as temperatures approach 37 C, the optimum temperature for fungal growth. To better characterize the influence of temperature on aflatoxin biosynthesis, we monitored the accumulation of aflatoxin and the expression of more than 5000 genes in Aspergillus flavus at 28 C and 37 C. A total of 144 genes were expressed differentially (P < 0.001) between the two temperatures. Among the 103 genes more highly expressed at 28 C, approximately 25% were involved in secondary metabolism and about 30% were classified as hypothetical. Genes encoding a catalase and superoxide dismutase were among those more highly expressed at 37 C. As anticipated we also found that all the aflatoxin biosynthetic genes were much more highly expressed at 28 C relative to 37 C. To our surprise expression of the pathway regulatory genes aflR and aflS, as well as aflR antisense, did not differ between the two temperatures. These data indicate that the failure of A. flavus to produce aflatoxin at 37 C is not due to lack of transcription of aflR or aflS. One explanation is that AFLR is nonfunctional at high temperatures. Regardless, the factor(s) sensing the elevated temperatures must be acute. When aflatoxin-producing cultures are transferred to 37 C they immediately stop producing aflatoxin. [ABSTRACT FROM AUTHOR]

Abstract: Aflatoxins are toxic secondary metabolites produced by Aspergillus flavus, Aspergillus parasiticus and Aspergillus nomius. A regulatory gene, aflR, is involved in regulation of aflatoxin biosynthesis and the sequence has been published. In this study, rapid assessment of aflatoxigenic fungi in food was accomplished using an indigenously designed primer pair for the aflatoxin regulatory gene aflR in polymerase chain reaction (PCR). Specificity was assayed in pure and mixed culture systems using DNA extracted from 28 different fungal strains as PCR template. Positive amplification was achieved only with DNA from aflatoxigenic A. flavus and A. parasiticus. The detection limit for mycelium and spores was determined as 0.05g and ≥100cfu, respectively. Specificity and sensitivity of PCR assay in groundnuts and maize for A. flavus was possible with as few as 100cfu/g. [Copyright &y& Elsevier]

Contamination of food and feedstuffs by Aspergillus species and their toxic metabolites is a serious problem as they have adverse effects on human and animal health. Hence, food contamination monitoring is an important activity, which gives information on the level and type of contamination. A PCR-based method of detection of Aspergillus species was developed in spiked samples of sterile maize flour. Gene-specific primers were designed to target aflR gene, and restriction fragment length polymorphism (RFLP) of the PCR product was done to differentiate Aspergillus flavus and Aspergillus parasiticus. Sterile maize flour was inoculated separately with A. flavus and A. parasiticus, each at several spore concentrations. Positive results were obtained only after 12-h incubation in enriched media, with extracts of maize inoculated with A. flavus (101 spores/g) and A. parasiticus (104 spores/g). PCR products were subjected to restriction endonuclease (HincII and PvuII) analysis to look for RFLPs. PCR-RFLP patterns obtained with these two enzymes showed enough differences to distinguish A. flavus and A. parasiticus. This approach of differentiating these two species would be simpler, less costly and quicker than conventional sequencing of PCR products. [Copyright &y& Elsevier]

Aspergillus sojae, which is believed to be a domesticated strain of Aspergillus parasiticus, contains all of the aflatoxin biosynthetic genes but is unable to produce aflatoxins and is generally recognized as safe (GRAS) for producing fermented foods. In A. parasiticus both aflR, the aflatoxin pathway-specific regulatory gene, and aflJ, a co-activator gene, are necessary for transcription of genes encoding the aflatoxin biosynthetic enzymes. A. sojae aflR differs from A. parasiticus aflR in that it encodes extra His and Ala, and has a pretermination defect that causes truncation of the carboxyl terminus of the predicted protein. A. sojae aflJ differs from A. parasiticus aflJ in that it encodes a predicted protein with Ser39 replaced by Ala and Ser283 replaced by Pro. Steady-state levels of aflatoxin biosynthetic gene transcripts of aflR, aflJ, pksA, nor1, ver1 and omtA in A. sojae as determined by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) were much lower than those of A. parasiticus. Yeast two-hybrid assays showed that the truncated A. sojae AFLR did not interact with AFLJ of A. sojae and A. parasiticus but that an A. sojae AFLR reverted to the putative ancestral form interacted normally with AFLJ of A. sojae and A. parasiticus. Deletion analysis showed that both amino- and carboxy-terminal regions of the A. sojae AFLJ were important for the R–J interaction. The truncated A. sojae AFLR thus not only was impaired in its ability to activate transcription of aflatoxin biosynthetic genes, but also was unable to interact with AFLJ, in A. parasiticus both of which are required for normal expression of the aflatoxin biosynthetic genes. Consequently, the lack of aflatoxin-producing ability of A. sojae resulted primarily from two defects in the regulatory mechanism responsible for gene transcription. [Copyright &y& Elsevier]

Senna (Cassia angustifolia Vahl.) is a medicinal crop with laxative properties, and it has significant demand in the global pharmaceutical market. Senna pods are highly susceptible to aflatoxin contamination, and the successful export of pods is hindered due to the regulatory limits of importing countries. The senna pod water activity (a w) from harvest to storage is the key factor determining AFB 1 accumulation. The temperature conditions from field to warehouse also interact with pod a w , which influences fungal growth and AFB 1 production. The determination of an ideal combination of a w and temperature led to the assessment of the critical control point for AFB 1 synthesis in senna. Hence, this study aimed to evaluate the influence of a w (0.99, 0.96, 0.93, 0.90, and 0.87 a w) and temperature (20, 28, and 37 °C) on fungal growth, gene expression (aflR and aflS), and AFB 1 production by A. flavus in senna agar medium. The fungus showed the longest lag time (7.7 days) at 20 °C with 0.87 a w. We observed that 0.96 a w (P < 0.01) was optimum for the diametric growth rate at 28 and 37 °C. However, the peak expression of regulatory genes (aflR and aflS) and the maximum AFB 1 production were observed only at 28 °C (0.96 a w). The highest growth rate occurred at 37 °C, which did not favor the expression of genes and AFB 1 production. However, at 28 °C, it positively correlated with gene expression and AFB 1 production. The suppressed expression of regulatory genes and a trace amount of aflatoxin B 1 were found at 20 °C with all the tested a w. In our experiments, the low a w (0.87 and 0.90 a w) suppressed the fungal growth, gene expression, and AFB 1 production of A. flavus at all of the tested temperatures (20, 28, and 37 °C). The rapid drying of senna pods with a low water activity (≤0.87 a w) and storage at low temperature (20 °C) are ideal conditions to avoid AFB 1 and ensure the quality of produce for export. • Temperature and a w are the key factors for aflatoxin contamination in senna pods. • flavus had the longest lag phase and slowest growth rate at 20 °C with 0.87 a w. • The optimum condition for AFB 1 production was found at 28 °C with 0.96 a w. • The fastest fungal growth observed at 37 °C was not correlated with higher AFB 1. • Storing pods at 20 °C with ≤0.87 a w is ideal to avoid aflatoxin contamination. [ABSTRACT FROM AUTHOR]

Regulation of aflatoxin (AF) biosynthesis likely involves a complex interplay of positive- and negative-acting factors that are affected by physiological cues responsive to internal and external stimuli. These factors, presumably, modulate the expression of the AF pathway-specific regulatory gene, aflR, whose product, AFLR, a zinc cluster transcription factor, then turns on or off the transcription of other AF genes. To determine if the AFLR carboxyl region (AFLRC) interacts with positive-or negative-acting proteins, we fused the Aspergillus parasiticus aflR carboxyl coding region( aflRC) to the promoter of A. parasiticusnitrite reductase gene ( niiA(p)::aflRC), and transformed it into A. parasiticus SRRC 2043. Transformants that contained two copies of niiA(p)::aflRC, one at the niaD locus and another at the aflR locus, over produced AF precursors independent of the nitrogen source. The higher copy number of the integrated niiA(p)::aflRC correlated with increased production of AF precursors by the transformants as well as increased expression of both aflRC and native aflR in potato dextrose broth and A & M medium. Since aflRC does not encode a DNA-binding domain, the expressed AFLRC should not bind to the promoters of AF pathway genes and affect transcription directly. The results are consistent with AFLRC titrating out a putative repressor that interacts with AFLR under different growth conditions and modulates AF biosynthesis. This interaction also indirectly affects sclerotial development. [ABSTRACT FROM AUTHOR]

Pistachio nuts are an important economic tree nut crop which is used directly or processed for many food-related activities. They can become colonized by mycotoxigenic spoilage fungi, especially Aspergillus flavus, mainly resulting in contamination with aflatoxins (AFs), especially aflatoxin B1 (AFB1). The prevailing climate in which these crops are grown changes as temperature and atmospheric CO2 levels increase, and episodes of extreme wet/dry cycles occur due to human industrial activity. The objectives of this study were to evaluate the effect of interacting Climate Change (CC)-related abiotic factors of temperature (35 vs. 37 °C), CO2 (400 vs. 1000 ppm), and water stress (0.98–0.93 water activity, aw) on (a) growth (b) aflD and aflR biosynthetic gene expression and (c) AFB1 production by two strains A. flavus (AB3, AB10) in vitro on milled pistachio-based media and when colonizing layers of shelled raw pistachio nuts. The A. flavus strains were resilient in terms of growth on pistachio-based media and the colonisation of pistachio nuts with no significant difference when exposed to the interacting three-way climate-related abiotic factors. However, in vitro studies showed that AFB1 production was significantly stimulated (p < 0.05), especially when exposed to 1000 ppm CO2 at 0.98–0.95 aw and 35 °C, and sometimes in the 37 °C treatment group at 0.98 aw. The relative expression of the structural aflD gene involved in AFB1 biosynthesis was decreased or only slightly increased, relative to the control conditions at elevated CO, regardless of the aw level examined. For the regulatory aflR gene expression, there was a significant (p < 0.05) increase in 1000 ppm CO2 and 37 °C for both strains, especially at 0.95 aw. The in situ colonization of pistachio nuts resulted in a significant (p < 0.05) stimulation of AFB1 production at 35 °C and 1000 ppm CO2 for both strains, especially at 0.98 aw. At 37 °C, AFB1 production was either decreased, in strain AB3, or remained similar, as in strain AB10, when exposed to 1000 ppm CO2. This suggests that CC factors may have a differential effect, depending on the interacting conditions of temperature, exposure to CO2 and the level of water stress on AFB1 production. [ABSTRACT FROM AUTHOR]