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After interruption or deletion of the photosystem II genes psbB, psbC, and psbD in the cyanobacterium Synechocystis sp. PCC 6803, thylakoids from such mutants were found to be depleted in a number of photosystem II proteins in addition to those for which the gene(s) had been inactivated. Transcript levels of photosystem II genes were measured and protein pulse-labeling was carried out to determine the reason for this effect. Transcripts of all photosystem II genes except the inactivated one(s) were found to be present in the various mutants. In certain cases, inactivation of one photosystem II gene led to overexpression of another. Protein pulse-labeling experiments using 35S-methionine, in which not only the rapidly turning over D1 protein but also D2, CP43, and CP47 appear to be preferentially labeled, showed that the mutants studied synthesize the D1 protein as well as other photosystem II proteins whose genes were not inactivated. The fact that, in the various mutants, photosystem II proteins for which the gene is not inactivated are synthesized but do not accumulate in the thylakoid indicates that the psbB, psbC, and psbD gene products are all required for a stable assembly of the photosystem II complex.

CONTENTS 2.1 Introduction 152.1.1 Ecology of Aspergillus fl avus 15 2.1.2 Aspergillus as a Plant Pathogen 16 2.1.3 Aspergillus as an Animal Pathogen 17 2.1.4 Aspergillus oryzae as an Ecotype of Aspergillus fl avus 172.2 Structural Genomics 18 2.2.1 Sequencing and Annotation 18 2.2.2 Physical Structure 18 2.2.3 Database 192.3 Comparative Genomics 19 2.3.1 Physical Structure 19 2.3.2 Unique Genes and Features 19 2.3.3 Population Analysis 192.4 Functional Genomics 20 2.4.1 Design of Affymetrix Arrays 20 2.4.2 Integrated Database for Functional Analysis 202.5 Summary and Prospects 21 Acknowledgments 21 References 21Aspergillus fl avus is a competitive saprobe, a plant pathogen, and an animal pathogen; thus, its ecology encompasses all of the known trophic phases of the genus Aspergillus. In addition, A. fl avus has a rich profi le of secondary metabolites, including several mycotoxins. Known mycotoxins produced by A. fl avus include cyclopiazonic acid, aspertoxin, afl atrem, aspergillic acid, and afl atoxins.1 Afl atoxins, which are both toxic and carcinogenic, accumulate in food and feeds. Because of health concerns related to ingestion of afl atoxin, a large research community has developed with the focus of understanding the population biology, ecology, and secondary metabolism in this fungus. As a result, the population structure and ecology of A. fl avus is better described than for any other Aspergillus species and afl atoxin bio synthesis is one of the best-characterized pathways of secondary metabolism in fi lamentous fungi. Couple this with the recently available genome sequence and whole genome DNA microarrays, andA. fl avus emerges as an appealing model organism to study the biology, ecology, and pathogenicity of the genus.

CONTENTS 14.1 Introduction 395 14.2 Genetics of Aspergillus flavus 39714.2.1 Aflatoxin Biosynthesis 398 14.2.2 Regulatory Elements in Aflatoxin Biosynthesis 399 14.2.3 Genomics of A. flavus 40214.2.3.1 Expressed Sequence Tag Technology 403 14.2.3.2 DNA Microarrays 403 14.2.3.3 Whole Genome Sequencing 40614.3 Strategies to Control Preharvest Aflatoxin Contamination 406 14.3.1 Biological Control 406 14.3.2 Enhancement of Host-Plant Resistance 40714.3.2.1 Plant Breeding Strategies 408 14.3.2.2 Genetic Engineering for Resistanceto Aspergillus flavus 413 14.4 Conclusion 417 References 418Mycotoxins are typically low molecular weight organic compounds produced by fungi and are not considered to be essential to maintaining the life of the fungal cell (reviewed in Bhatnagar et al., 2002). They are called ‘‘secondary metabolites’’ because they are not involved in obtaining energy or synthesizing structural components of the cell. The well-studied mycotoxins are the aflatoxins because they are highly toxic and carcinogenic compounds that frequently contaminate foods and feeds. These compounds(Figure 14.1) are produced by several species of fungi in the genus Aspergillus. Aspergillus flavus, the most common species causing crop contamination, is an opportunistic pathogen, which routinely produces aflatoxins B1 and B2. Aspergillus parasiticus produces two additional toxins, namely G1, and G2. Aflatoxin B1 is both the most common and the most toxic and, as a result, is the aflatoxin most stringently regulated because it is the major toxicosis threat. Presence of aflatoxins in human foods may cause chronic health effects including immune-system suppression, impaired childhood development, and cancer. Acute poisoning can result in death (Wild and Turner,2002; Gong et al., 2004). In developing countries, where detection and decontamination policies and practices are impractical, aflatoxin contamination is predominantly a food safety issue. Recently Kenya reported a minimum of 125 people killed by consuming aflatoxin-contaminated maize (http:==www.cdc.gov=mmwr=preview=mmwr.html=mm533a4.htm). In developed countries, however, aflatoxin remains an economic problem because the contamination is most frequently recognized due to loss in crop value (estimated to be as high as $300 million in direct cost in the United States alone) resulting from strict government regulations on maximum permissible levels in crops and crop-related products (Cotty, 2001; Richard and Payne, 2003; Wu, 2004). In crops intended for human consumption, maximum permitted aflatoxin levels range from 2 ppb aflatoxin B1 (4 ppb total aflatoxins) in the European Union to 20 ppb total aflatoxins in the United States. Small proportions of crop aflatoxin content are transferred from feed to milk (aflatoxin M1) causing even food crops to have stringent regulations for use at dairies (van Egmond, 2004; Wu, 2004). Aflatoxin contamination occurrence frustrates not only the agricultural community but also all those that handle, market, or utilize crops from highly vulnerable regions. The crop may have no visible symptoms, and yet contain enough aflatoxins to either severely reduce value or completely eliminate available markets. The same fungal isolates may cause contamination of several crops grown in a given area, such as cottonseed, corn, peanuts, and tree nuts.

Aflatoxins (AFs) are carcinogenic secondary metabolites of Aspergillus parasiticus. In previous studies, non-toxigenic A. parasiticus sec- (for secondary metabolism negative) variants were generated through serial transfer of mycelia from their toxigenic sec+ (for secondary metabolism positive) parents for genetic and physiological analysis for understanding regulation of AF biosynthesis. Previous studies have shown no difference in the DNA sequence of aflR, a positive regulator of AF production, in the sec+ and sec- strains. In this study, AflJ, another positive regulator of AF production, laeA, a global regulator of secondary metabolism, and the intergenic region between aflR and aflJ, were analysed to determine if they play a role in establishment of the sec- phenotype. The study showed that while this sequence identity extended to the aflJ as well as the aflJ-aflR intergenic region, expression of aflR in the sec- strain was several fold lower than that observed in the sec+ strain, while aflJ expression was barely detectable in the sec- strain. Western blot analysis indicated that despite AflR protein being present in the sec- strain, no toxin production resulted. Introduction of a second copy of aflR into the sec- strain increased aflR expression, but did not restore AF production. Lastly, reverse transcription-PCR analysis revealed that laeA was expressed in both sec+ and sec- strains. These results suggest that although aflR, aflJ and laeA are necessary for AF production, they are not sufficient. We propose that the aflR and aflJ expression may be regulated by element(s) downstream from laeA or from pathways not influenced by laeA.

Aflatoxins are toxic and carcinogenic polyketide metabolites produced by certain fungal species, including Aspergillus flavus and A. parasiticus. Many internal and external factors, such as nutrition and environment affect aflatoxin biosynthesis; therefore, we analyzed the transcriptome of A. flavus using expressed sequence tags (ESTs) from a normalized cDNA expression library constructed from mycelia harvested under several conditions. A total of 7218 unique ESTs were identified from 26,110 sequenced cDNA clones. Functional classifications were assigned to these ESTs and genes, potentially involved in the aflatoxin contamination process, were identified. Based on this EST sequence information, a genomic DNA amplicon microarray was constructed at The Institute for Genomic Research (TIGR). To identify potential regulatory networks controlling aflatoxin contamination in food and feeds, gene expression profiles in aflatoxin-supportive media versus non-aflatoxin-supportive media were evaluated in A. flavus and A. parasiticus. Genes consistently expressed in several aflatoxin-supportive media are reported.

Caffeic acid (3,4-dihydroxycinnamic acid, 12 mM) added to a fat-based growth medium reduces > 95% of aflatoxin production by Aspergillus flavus NRRL 3357, without affecting fungal growth. Microarray analysis of caffeic acid-treated A. flavus indicated expression of almost all genes in the aflatoxin biosynthetic cluster were down-regulated, ranging from a log 2 ratio of caffeic acid treated and untreated of − 1.12 (medium) to − 3.13 (high). The only exceptions were genes norB and the aflatoxin pathway regulator-gene, aflJ , which showed low expression levels in both treated and control fungi. The secondary metabolism regulator-gene, laeA , also showed little change in expression levels between the fungal cohorts. Alternatively, expression of genes in metabolic pathways (i.e., amino acid biosynthesis, metabolism of aromatic compounds, etc.) increased (log 2 ratio > 1.5). The most notable up-regulation of A. flavus expression occurred in four genes that are orthologs of the Saccharomyces cerevisiae AHP1 family of genes. These genes encode alkyl hydroperoxide reductases that detoxify organic peroxides. These increases ranged from a log 2 ratio of 1.08 to 2.65 (moderate to high), according to real-time quantitative reverse transcription-PCR (qRT-PCR) assays. Based on responses of S. cerevisiae gene deletion mutants involved in oxidative stress response, caffeic, chlorogenic, gallic and ascorbic acids were potent antioxidants under oxidative stress induced by organic peroxides, tert -butyl and cumene hydroperoxides. Differential hypersensitivity to these peroxides and hydrogen peroxide occurred among different mutants in addition to their ability to recover with different antioxidants. These findings suggest antioxidants may trigger induction of genes encoding alkyl hydroperoxide reductases in A. flavus. The possibilities that induction of these genes protects the fungus from oxidizing agents (e.g., lipoperoxides, reactive oxygen species, etc.) produced during host-plant infection and this detoxification attenuates upstream signals triggering aflatoxigenesis are discussed.

The aflatoxin-producing fungi, Aspergillus flavus and A. parasiticus, form structures called sclerotia that allow for survival under adverse conditions. Deletion of the veA gene in A. flavus and A. parasiticus blocks production of aflatoxin as well as sclerotial formation. We used microarray technology to identify genes differentially expressed in wild-type veA and veA mutant strains that could be involved in aflatoxin production and sclerotial development in A. flavus. The DNA microarray analysis revealed 684 genes whose expression changed significantly over time; 136 of these were differentially expressed between the two strains including 27 genes that demonstrated a significant difference in expression both between strains and over time. A group of 115 genes showed greater expression in the wild-type than in the veA mutant strain. We identified a subgroup of veA-dependent genes that exhibited time-dependent expression profiles similar to those of known aflatoxin biosynthetic genes or that were candidates for involvement in sclerotial production in the wild type.

Aflatoxins are polyketide-derived, toxic, and carcinogenic secondary metabolites produced primarily by two fungal species, Aspergillus flavus and A. parasiticus, on crops such as corn, peanuts, cottonseed, and treenuts. Regulatory guidelines issued by the U.S. Food and Drug Administration (FDA) prevent sale of commodities if contamination by these toxins exceeds certain levels. The biosynthesis of these toxins has been extensively studied. About 15 stable precursors have been identified. The genes involved in encoding the proteins required for the oxidative and regulatory steps in the biosynthesis are clustered in a 70 kb portion of chromosome 3 in the A. flavus genome. With the characterization of the gene cluster, new insights into the cellular processes that govern the genes involved in aflatoxin biosynthesis have been revealed, but the signaling processes that turn on aflatoxin biosynthesis during fungal contamination of crops are still not well understood. New molecular technologies, such as gene microarray analyses, quantitative polymerase chain reaction (PCR), and chromatin immunoprecipitation are being used to understand how physiological stress, environmental and soil conditions, receptivity of the plant, and fungal virulence lead to episodic outbreaks of aflatoxin contamination in certain commercially important crops. With this fundamental understanding, we will be better able to design improved non-aflatoxigenic biocompetitive Aspergillus strains and develop inhibitors of aflatoxin production (native to affected crops or otherwise) amenable to agricultural application for enhancing host-resistance against fungal invasion or toxin production. Comparisons of aflatoxin-producing species with other fungal species that retain some of the genes required for aflatoxin formation is expected to provide insight into the evolution of the aflatoxin gene cluster, and its role in fungal physiology. Therefore, information on how and why the fungus makes the toxin will be valuable for developing an effective and lasting strategy for control of aflatoxin contamination.

Aspergillus fumigatus is a filamentous fungal saprophyte that is ubiquitous in the environment. It is also a human pathogen and induces allergenic response, negatively impacting health care and associated costs significantly around the world. Much of the basic biology of this organism is only poorly understood, but the recent completion and publication of its genome sequence provides an excellent tool for researchers to gain insight into these processes. In this review we will summarize some of the more salient features revealed by analysis of the genome, including the search for candidate pathogenicity genes and the switch to a pathogenic lifestyle, allergen proteins, DNA repair, secondary metabolite gene clusters that produce compounds both useful and toxic, a theoretical capability of this asexual organism to reproduce sexually, signalling, and transcription. A. fumigatus was compared with the food biotechnology fungus Aspergillus oryzae and sexual fungus Aspergillus nidulans, as well as other fungi, in an attempt to discern key differences between these organisms.

Aspergillus flavus is an imperfect filamentous fungus that is an opportunistic pathogen causing invasive and non-invasive aspergillosis in humans, animals, and insects. It also causes allergic reactions in humans. A. flavus infects agricultural crops and stored grains and produces the most toxic and potent carcinogic metabolites such as aflatoxins and other mycotoxins. Breakthroughs in A. flavus genomics may lead to improvement in human health, food safety, and agricultural economy. The availability of A. flavus genomic data marks a new era in research for fungal biology, medical mycology, agricultural ecology, pathogenicity, mycotoxin biosynthesis, and evolution. The availability of whole genome microarrays has equipped scientists with a new powerful tool for studying gene expression under specific conditions. They can be used to identify genes responsible for mycotoxin biosynthesis and for fungal infection in humans, animals and plants. A. flavus genomics is expected to advance the development of therapeutic drugs and to provide information for devising strategies in controlling diseases of humans and other animals. Further, it will provide vital clues for engineering commercial crops resistant to fungal infection by incorporating antifungal genes that may prevent aflatoxin contamination of agricultural harvest.

The genome of Aspergillus oryzae, a fungus important for the production of traditional fermented foods and beverages in Japan, has been sequenced. The ability to secrete large amounts of proteins and the development of a transformation system1 have facilitated the use of A. oryzae in modern biotechnology2-4. Although both A. oryzae and Aspergillus flavus belong to the section Flavi of the subgenus Circumdati of Aspergillus, A. oryzae, unlike A. flavus, does not produce aflatoxin, and its long history of use in the food industry has proved its safety. Here we show that the 37-megabase (Mb) genome of A. oryzae contains 12,074 genes and is expanded by 7-9 Mb in comparison with the genomes of Aspergillus nidulans5 and Aspergillus fumigatus6. Comparison of the three aspergilli species revealed the presence of syntenic blocks and A. oryzae-specific blocks (lacking synteny with A. nidulans and A. fumigatus) in a mosaic manner throughout the genome of A. oryzae. The blocks of A. oryzae-specific sequence are enriched for genes involved in metabolism, particularly those for the synthesis of secondary metabolites. Specific expansion of genes for secretory hydrolytic enzymes, amino acid metabolism and amino acid/sugar uptake transporters supports the idea that A. oryzae is an ideal microorganism for fermentation. © 2005 Nature Publishing Group.

K.C. EHRLICH, J. YU AND P.J. COTTY. 2005. Aims: To compare the biosynthetic gene cluster sequences of the main aflatoxin (AF)-producing Aspergillus species. Methods and Results: Sequencing was on fosmid clones selected by homology to Aspergillus parasiticus sequence. Alignments revealed that gene order is conserved among AF gene clusters of Aspergillus nomius, A. parasiticus, two sclerotial morphotypes of Aspergillus flavus, and an unnamed Aspergillus sp. Phylogenetic relationships were established using the maximum likelihood method implemented in PAUP. Based on the Eurotiomycete/ Sordariomycete divergence time, the A. flavus-type cluster has been maintained for at least 25 million years. Such conservation of the genes and gene order reflects strong selective constraints on rearrangement. Phylogenetic comparison of individual genes in the cluster indicated that ver-1, which has homology to a melanin biosynthesis gene, experienced selective forces distinct from the other pathway genes. Sequences upstream of the polyketide synthase-encoding gene vary among the species, but a four-gene sugar utilization cluster at the distal end is conserved, indicating a functional relationship between the two adjacent clusters. Conclusions: The high conservation of cluster components needed for AF production suggests there is an adaptive value for AFs in character-shaping niches important to those taxa. Significance and Impact of the Study: This is the first comparison of the complete nucleotide sequences of gene clusters harbouring the AF biosynthesis genes of the main AF-producing species. Such a comparison will aid in understanding how AF biosynthesis is regulated in experimental and natural environments.

Aflatoxins, produced primarily by Aspergillus flavus and A. parasiticus, are among the most toxic and carcinogenic naturally occurring compounds. In an attempt to identify genes potentially involved in aflatoxin contamination of crops, and to better understand the biology of A. flavus, a large scale sequencing of A. flavus expressed sequence tags (EST) was conducted. The 5' ends of 26,110 cDNA clones from a normalized cDNA expression library were sequenced. After annotation, a total of 7218 unique ESTs in A. flavus were assembled into 3749 tentative concensus sequences and 3469 singleton sequences. The functional classifications of the genes or Gene Ontology (GO) terms were assigned to these ESTs. Genes potentially involved in the aflatoxin contamination process were identified in the ESTs sequenced. These include the aflatoxin biosynthetic pathway, signal transduction, global regulation, pathogenicity of the fungus, and stress response.

Aflatoxins, a group of polyketide-derived furanocoumarins (Fig. [1][1]), are the most toxic and carcinogenic compounds among the known mycotoxins. Among the at least 16 structurally related aflatoxins characterized, however, there are only four major aflatoxins, B1, B2, G1, and G2 (AFB1, AFG1, AFB2

An 82-kb Aspergillus parasiticus genomic DNA region representing the completed sequence of the well-organized aflatoxin pathway gene cluster has been sequenced and annotated. In addition to the 19 reported and characterized aflatoxin pathway genes and the four sugar utilization genes in this cluster, we report here the identification of six newly identified genes which are putatively involved in aflatoxin formation. The function of these genes, the cluster organization and its significance in gene expression are discussed.

Summary A. flavus is the most common cause of aflatoxin contamination in pre-harvest filed crops and post-harvest grains. It is a characteristic “opportunistic” plant pathogen that infects corn, cotton, peanuts, and treenuts, and contaminates them with aflatoxins. There are no effective control strategies to prevent aflatoxin accumulation in the field when conditions are favorable for fungal growth. The mode of action and metabolism of aflatoxin biosynthesis have been extensively studied. Chemical binding of liver enzyme-activated aflatoxin molecules to animal DNA, causing mutations and possible carcinogenesis, has been demonstrated. The chemistry, biochemistry, molecular biology, and synthesis of aflatoxins B 1 , and B 2 , and the transcriptional activation of the genes involved are understood in significant detail. We have discovered an aflatoxin pathway gene cluster, a sugar utilization gene cluster, and a nitrogen pathway gene cluster. However, the mechanisms of aflatoxin formation in terms of global regulation, pathogenicity of the fungus, and crop-fungus interactions are poorly understood. A large research community has developed focused on understanding the biology of the fungus, and the biosynthesis of aflatoxins, with the goal of developing novel control strategies. A. flavus genomics will provide a powerful tool for identification of genes of interest. Microarrays containing A. flavus gene fragments can be used to study gene expression profiles under diverse conditions. Aspergillus , particularly, the species in the flavus group, has been recognized as a model system for studying fungal biology and pathogeneicity in solving food safety related issues. Identification and functional elucidation of those genes that are responsible for aflatoxin formation, regulation, signal transduction, pathogenicity, and the environmental effects on aflatoxin production by the fungus will provide vital information for devising new strategies to eliminate pre-harvest aflatoxin contamination, resulting in a safer, economically viable food and feed supply.

Aflatoxins are toxic and extremely carcinogenic natural secondary metabolites produced primarily by the fungi Aspergillus flavus and Aspergillus parasiticus. The biosynthesis of aflatoxins is a complex process involving multi-enzymatic reactions. Genetic studies of the molecular mechanism of aflatoxin B1 biosynthesis have identified an aflatoxin pathway gene cluster of 70 kilobase pairs in length consisting of at least 24 identified structural genes including a positive regulatory gene as transcription activator. The structural genes encode cytochrome P450 monooxygenases, dehydrogenases, oxidases, methyltransferases, a polyketide synthase and two unique fatty acid synthases. The aflatoxin biosynthesis and its genetic regulation are discussed in this review. The current knowledge of the relationship between fungal development and secondary metabolism is also summarized.

Within the 80 kb aflatoxin pathway gene cluster characterized earlier, and between adhA and norA genes, we have identified an estA gene encoding an esterase from wild type strain Aspergillus parasiticus SRRC 143. The 1,500 bp genomic DNA and 945 bp cDNA sequences were determined for estA. Outside of the aflatoxin pathway gene cluster, an additional copy of the estA gene (named estA2) was also cloned from the same A. parasiticus strain. Comparison of the estA and estA2 sequences showed 9 substitutions within the 314 amino acid residues of their gene products, and no apparent defect was identified in the estA2. The estA gene is a homolog of the stcI gene identified in A. nidulans involved in the biosynthesis of sterigmatocystin and dihydro-sterigmatocystin for the conversion of versiconal hemiacetal acetate to versiconal. Reverse-transcriptase polymerase chain reaction (RT-PCR) experiments demonstrated that the estA is constitutively expressed. And only this estA gene, which is located within the aflatoxin pathway gene cluster, is expressed; no expression of the estA2 gene was detected under both aflatoxin conducive and non-conducive conditions. Possible reasons for the preferential expression of the estA over the estA2 gene have been discussed.

Aspergillus sojae belongs to the Aspergillus section Flavi but does not produce aflatoxins. The functionality of the A. sojae aflR gene ( aflRs ) was examined by transforming it into an Δ aflR strain of A. parasiticus , derived from a nitrate-nonutilizing, versicolorin A (VERA)-accumulating strain. The A. parasiticus aflR gene ( aflRp ) transformants produced VERA, but the aflRs transformants did not. Even when aflRs was placed under the control of the amylase gene ( amyB ) promoter of Aspergillus oryzae , the amy ( p ):: aflRs transformants did not produce VERA. A chimeric construct containing the aflRs promoter plus the aflRs N- and aflRp C-terminal coding regions could restore VERA production, but a construct containing the aflRp promoter plus the aflRp N- and aflRs C-terminal coding regions could not. These results show that the A. sojae aflR promoter is functional in A. parasiticus and that the HAHA motif does not affect the function of the resulting hybrid AflR. We conclude that the lack of aflatoxin production by A. sojae can be attributed, at least partially, to the premature termination defect in aflRs , which deletes the C-terminal transcription activation domain that is critical for the expression of aflatoxin biosynthetic genes.

Mycotoxins are low-molecular-weight secondary metabolites of fungi. The most significant mycotoxins are contaminants of agricultural commodities, foods and feeds. Fungi that produce these toxins do so both prior to harvest and during storage. Although contamination of commodities by toxigenic fungi occurs frequently in areas with a hot and humid climate (i.e. conditions favorable for fungal growth), they can also be found in temperate conditions. Production of mycotoxins is dependent upon the type of producing fungus and environmental conditions such as the substrate, water activity (moisture and relative humidity), duration of exposure to stress conditions and microbial, insect or other animal interactions. Although outbreaks of mycotoxicoses in humans have been documented, several of these have not been well characterized, neither has a direct correlation between the mycotoxin and resulting toxic effect been well established in vivo. Even though the specific modes of action of most of the toxins are not well established, acute and chronic effects in prokaryotic and eukaryotic systems, including humans have been reported. The toxicity of the mycotoxins varies considerably with the toxin, the animal species exposed to it, and the extent of exposure, age and nutritional status. Most of the toxic effects of mycotoxins are limited to specific organs, but several mycotoxins affect many organs. Induction of cancer by some mycotoxins is a major concern as a chronic effect of these toxins. It is nearly impossible to eliminate mycotoxins from the foods and feed in spite of the regulatory efforts at the national and international levels to remove the contaminated commodities. This is because mycotoxins are highly stable compounds, the producing fungi are ubiquitous, and food contamination can occur both before and after harvest. Nevertheless, good farm management practices and adequate storage facilities minimize the toxin contamination problems. Current research is designed to develop natural biocontrol competitive fungi and to enhance host resistance against fungal growth or toxin production. These efforts could prevent toxin formation entirely. Rigorous programs for reducing the risk of human and animal exposure to contaminated foods and feed also include economically feasible and safe detoxification processes and dietary modifications. Although risk assessment has been made for some mycotoxins, additional, systematic epidemological data for human exposure is needed for establishing toxicological parameters for mycotoxins and the safe dose for humans. It is unreasonable to expect complete elimination of the mycotoxin problem. But multiple approaches will be needed to minimize the economic impact of the toxins on the entire agriculture industry and their harmfulness to human and animal health.

Mycotoxins are low-molecular-weight secondary metabolites of fungi. The most significant mycotoxins are contaminants of agricultural commodities, foods and feeds. Fungi that produce these toxins do so both prior to harvest and during storage. Although contamination of commodities by toxigenic fungi occurs frequently in areas with a hot and humid climate (i.e. conditions favorable for fungal growth), they can also be found in temperate conditions. Production of mycotoxins is dependent upon the type of producing fungus and environmental conditions such as the substrate, water activity (moisture and relative humidity), duration of exposure to stress conditions and microbial, insect or other animal interactions. Although outbreaks of mycotoxicoses in humans have been documented, several of these have not been well characterized, neither has a direct correlation between the mycotoxin and resulting toxic effect been well established in vivo. Even though the specific modes of action of most of the toxins are not well established, acute and chronic effects in prokaryotic and eukaryotic systems, including humans have been reported. The toxicity of the mycotoxins varies considerably with the toxin, the animal species exposed to it, and the extent of exposure, age and nutritional status. Most of the toxic effects of mycotoxins are limited to specific organs, but several mycotoxins affect many organs. Induction of cancer by some mycotoxins is a major concern as a chronic effect of these toxins. It is nearly impossible to eliminate mycotoxins from the foods and feed in spite of the regulatory efforts at the national and international levels to remove the contaminated commodities. This is because mycotoxins are highly stable compounds, the producing fungi are ubiquitous, and food contamination can occur both before and after harvest. Nevertheless, good farm management practices and adequate storage facilities minimize the toxin contamination problems. Current research is designed to develop natural biocontrol competitive fungi and to enhance host resistance against fungal growth or toxin production. These efforts could prevent toxin formation entirely. Rigorous programs for reducing the risk of human and animal exposure to contaminated foods and feed also include economically feasible and safe detoxification processes and dietary modifications. Although risk assessment has been made for some mycotoxins, additional, systematic epidemological data for human exposure is needed for establishing toxicological parameters for mycotoxins and the safe dose for humans. It is unreasonable to expect complete elimination of the mycotoxin problem. But multiple approaches will be needed to minimize the economic impact of the toxins on the entire agriculture industry and their harmfulness to human and animal health.

Penicillium crustosum Thom (1930) causes blue mold on pome fruits and is also regularly found on cheese, nuts, and soil (1,3). The fungus produces a wide range of mycotoxins such as penitrem A, roquefortine C, terrestric acid, and cyclopenol, which impact human health (1). In January and February 2013, 20 decayed apples, ‘Golden Delicious’ and ‘Jonagold’ (Malus × domestica Borkh.) with blue mold symptoms were collected from cold storages in Svilajnac and Bela Crkva, Serbia. Decayed areas were light to medium brown with blue green sporulation on the surface of the lesion. Decayed tissue was soft and watery with a sharp margin between the diseased and healthy areas. One isolate from each cultivar was designated JP2 (‘Golden Delicious’) and JBC7 (‘Jonagold’) and further characterized. Conidiophores of both isolates were terverticillate, stipes were septate with rough walls, and phialides were ampulliform. Conidia were smooth, borne in columns, and were spherical to subglobose. Conidial dimensions for JP2 were 3.2 to 4.56 (3.73) × 2.64 to 4.3 (3.32) μm and for JBC7 were 3.1 to 4.46 (3.65) × 2.81 to 4.27 (3.31) μm (n = 50). The isolates were cultured on Czapek yeast autolysate agar (CYA), malt extract agar (MEA), and yeast extract sucrose agar (YES) media and incubated at 25°C for 7 days. Mycelia were white with heavy sporulation yielding grayish green colonies on all media. Colonies were radially sulcate and velutinous, with clear exudate, and produced a yellow to orange reverse on CYA and YES. On MEA, colonies were plane, low, and mycelia subsurface with conidia having a dry powdery appearance. Crusts of conidial masses formed after 10 or more days. No growth was observed on CYA when these isolates were incubated at 37°C. Both isolates were identified as P. crustosum Thom using morphological characters according to (2) and (1). Species level identification was confirmed by isolating genomic DNA followed by amplification of the β-tubulin locus using gene specific primers via conventional PCR (4). MegaBLAST analysis of the 2X consensus nucleotide sequences revealed that JP2 and JBC7 (GenBank KJ433984 and 85) were 99% identical to P. crustosum culture collection isolate IBT 21518 (JN112030.1). Koch's postulates were examined using two apple cvs. Idared and Kolacara. Ten fruit per cultivar per isolate were inoculated on two sides of each fruit; 20 fruit were used as water-only inoculated controls. Fruit were washed with soap and water, surface sanitized with 70% ethanol, and placed into polyethylene boxes. Using a finishing nail, 4-mm wounds were created and inoculated with 50 μl of a 3 × 105/ml conidial suspension or Tween-treated sterile distilled water. Boxes with inoculated and control fruit were stored at 25°C for 10 days. The inoculated fruit developed small, soft, watery lesions, which enlarged into decayed areas with defined edges and abundant sporulation on the surface. Symptoms were identical to the original ones, while the control fruit remained symptomless. The fungus was re-isolated from infected tissue and showed the same morphological characteristics as the original isolates, thus completing Koch's postulates. Blue mold occurs during long term storage of apples and is predominantly caused by P. expansum. This is the first report of P. crustosum causing postharvest blue mold decay on apple fruit obtained from storage in Serbia and indicates that P. crustosum is an emerging pathogen for the Serbian pome fruit growing and packing industry. References: (1) J. C. Frisvad and R. A. Samson. Stud. Mycol. 49:1, 2004. (2) J. I. Pitt and A. D. Hocking. Fungi and Food Spoilage, 239. Springer, 2009. (3) P. G. Sanderson and R. A. Spotts. Phytopathology 85:103. 1995. (4) P. L. Sholberg et al. Postharvest Biol. Technol. 36:41, 2005.

Two routes for the conversion of 5′-hydroxyaverantin (HAVN) to averufin (AVF) in the synthesis of aflatoxin have been proposed. One involves the dehydration of HAVN to the lactone averufanin (AVNN), which is then oxidized to AVF. Another requires dehydrogenation of HAVN to 5′-ketoaverantin, the open-chain form of AVF, which then cyclizes spontaneously to AVF. We isolated a gene, adhA , from the aflatoxin gene cluster of Aspergillus parasiticus SU-1. The deduced ADHA amino acid sequence contained two conserved motifs found in short-chain alcohol dehydrogenases—a glycine-rich loop (GXXXGXG) that is necessary for interaction with NAD + -NADP + , and the motif YXXXK, which is found at the active site. A. parasiticus SU-1, which produces aflatoxins, has two copies of adhA ( adhA1 ), whereas A. parasiticus SRRC 2043, a strain that accumulates O -methylsterigmatocystin (OMST), has only one copy. Disruption of adhA in SRRC 2043 resulted in a strain that accumulates predominantly HAVN. This result suggests that ADHA is involved in the dehydrogenation of HAVN to AVF. Those adhA disruptants that still made small amounts of OMST also accumulated other metabolites, including AVNN, after prolonged culture.

The identification of overlapping cosmids resulted in the discovery of the aflatoxin biosynthetic pathway gene cluster in Aspergillus flavus and A. parasiticus. This finding led to the cloning and characterization of one regulatory and 16 structural genes involved in aflatoxin biosynthesis, including the most recent report on the gene, ordA, which has been identified to be involved in the formation of four aflatoxins (B1, B2, G1 and G2). However, these genes do not account for all the identified chemical/biochemical steps in aflatoxin synthesis and efforts are underway to identify the genes controlling the other steps. We are also attempting to define the outer boundaries of the aflatoxin pathway gene cluster in the Aspergillus genome. For this goal, we extended sequencing in both directions from the existing (60 kb) aflatoxin pathway gene cluster, beyond the pksA gene at one end and the omtA gene at the other. Within the 25-kb genomic DNA sequence determined at the omtA end of the cluster, several new gene sequences were identified. The recently reported genes, vbs and ordA, were found within this 25-kb region. Two additional genes were also found in this region, a cytochrome P450 monooxygenase encoding gene, tentatively named cypX, and a monooxygenase encoding gene, tentatively named moxY, and these are also reported in this study. The sequence beyond these genes showed a 5-kb non-coding region of DNA followed by the presence of a cluster of genes probably involved in sugar metabolism. Northern blot analysis and reverse transcriptase-polymerase chain reaction (RT-PCR) studies demonstrated that the genes, cypX and moxY, are expressed concurrently with genes involved in aflatoxin biosynthesis. Therefore, the two putative aflatoxin pathway genes cypX and moxY followed by a 5-kb non-coding region of DNA define one end of the boundary of the aflatoxin pathway gene cluster in A. parasiticus.

The biosynthesis of aflatoxins (B(1), G(1), B(2), and G(2)) is a multi-enzyme process controlled genetically by over 20 genes. In this study, we report the identification and characterization of the avfA gene, which was found to be involved in the conversion of averufin (AVF) to versiconal hemiacetal acetate (VHA), in Aspergillus parasiticus and A. flavus; a copy of avfA gene was also cloned from a non-aflatoxin producing strain A. sojae. Complementation of an averufin-accumulating, non-aflatoxigenic mutant strain of A. parasiticus, SRRC 165, with the avfA gene cloned from A. flavus, restored the ability of the mutant to convert AVF to VHA and to produce aflatoxins B(1), G(1), B(2), and G(2). Sequence analysis revealed that a single amino acid replacement from aspartic acid to asparagine disabled the function of the enzyme in the mutant strain SRRC 165. The A. parasiticus avfA was identified to be a homolog of previously sequenced, but functionally unassigned transcript, stcO, in A. nidulans based on sequence homology at both nucleotide (57%) and amino acid (55%) levels. In addition to avfA, another aflatoxin pathway gene, omtB, encoding for an O-methyltransferase involved in the conversion of demethylsterigmatocystin (DMST) to sterigmatocystin (ST) and dihydrodemethylsterigmatocystin (DHDMST) to dihydrosterigmatocystin (DHST), was cloned from A. parasiticus, A. flavus, and A. sojae. The omtB gene was found to be highly homologous to stcP from A. nidulans, which has been reported earlier to be involved in a similar enzymatic step for the sterigmatocystin formation in that species. RT-PCR data demonstrated that both the avfA and avfA1 as well as omtB genes in A. parasiticus were expressed only in the aflatoxin-conducive medium. An analysis of the degrees of homology for the two reported genes between the Aspergillus species A. parasiticus, A. flavus, A. nidulans and A. sojae was conducted.

O -methyltransferase (OMT) is one of the key enzymes in aflatoxin (AF) biosynthesis in the fungi, Aspergillus flavus (Af) and A. parasiticus (Ap) . Genomic DNA clones containing the omtA genes from Ap strain SRRC 143 and Afstrain CRA01-2B were sequenced. Comparison of the genomic DNA sequences with the cDNA of this Ap gene revealed the presence of four introns ranging from 52 to 60 bp in length in both species; the region encoding the putative Sadenosylmethionine-binding motif was located between the third and fourth introns. The coding sequence of omtA from Ap strain SRRC 143 demonstrated a greater than 97% sequence identity with that from Af strain CRA01-2B, within the coding region.

Aflatoxins comprise a group of polyketide-derived carcinogenic mycotoxins produced by Aspergillus parasiticus and Aspergillus flavus. By transformation with a disruption construct, pXX, we disrupted the aflatoxin pathway in A. parasiticus SRRC 2043, resulting in the inability of this strain to produce aflatoxin intermediates as well as a major yellow pigment in the transformants. The disruption was attributed to a single-crossover, homologous integration event between pXX and the recipient A. parasiticus genome at a specific locus, designated pksA. Sequence analysis suggest that pksA is a homolog of the Aspergillus nidulans wA gene, a polyketide synthase gene involved in conidial wall pigment biosynthesis. The conserved beta-ketoacyl synthase, acyltransferase and acyl carrier-protein domains were present in the deduced amino acid sequence of the pksA product. No beta-ketoacyl reductase and enoyl reductase domains were found, suggesting that pksA does not encode catalytic activities for processing beta-carbon similar to those required for long chain fatty acid synthesis. The pksA gene is located in the aflatoxin pathway gene cluster and is linked to the nor-1 gene, an aflatoxin pathway gene required for converting norsolorinic acid to averantin. These two genes are divergently transcribed from a 1.5 kb intergenic region. We propose that pksA is a polyketide synthase gene required for the early steps of aflatoxin biosynthesis.

Aflatoxins are toxic and carcinogenic secondary metabolites produced by the fungi Aspergillus flavus and A. parasiticus. Aflatoxins are synthesized by condensation of acetate units; their synthesis is estimated to involve at least 16 different enzymes. In this study we have shown that at least nine genes involved in the aflatoxin biosynthetic pathway are located within a 60-kb DNA fragment. Four of these genes, nor-1, aflR, ver-1, and omtA (previously named omt-1), have been cloned in A. flavus and A. parasiticus. In addition, five other genes, pksA, uvm8, aad, ord-1, and ord-2 have been recently cloned in A. parasiticus. The pksA, aad, and uvm8 genes exhibit sequence homologies to polyketide synthase, aryl-alcohol dehydrogenase, and fatty acid synthase genes, respectively. The cDNA sequences of ord-1 and ord-2 genes, which may be involved in later steps of aflatoxin biosynthesis, have been determined; the ord-1 gene product exhibits homology to cytochrome P-450-type enzymes. By characterizing the overlapping regions of the DNA inserts in different cosmid and lambda DNA clones, we have determined the order of these aflatoxin pathway genes within this 60-kb DNA region to be pksA, nor-1, uvm8, aflR, aad, ver-1, ord-1, ord-2, and omtA in A. parasiticus and nor-1, aflR, ver-1, ord-1, ord-2, and omtA in A. flavus. The order is related to the order in enzymatic steps required for aflatoxin biosynthesis. The physical distances (in kilobase pairs) and the directions of transcription of these genes have been determined for both aflatoxigenic species.

The aflR gene from Aspergillus parasiticus and Aspergillus flavus may be involved in the regulation of aflatoxin biosynthesis. The aflR gene product, AFLR, possesses a GAL4-type binuclear zinc finger DNA-binding domain. A transformant, SU1-N3 (pHSP), containing an additional copy of aflR, showed increased transcription of aflR and the aflatoxin pathway structural genes, nor-1, ver-1, and omt-1, when cells were grown in nitrate medium, which normally suppresses aflatoxin production. Electrophoretic mobility shift assays showed that the recombinant protein containing the DNA-binding domain, AFLR1, bound specifically to the palindromic sequence, TTAGGCCTAA, 120 bp upstream of the AFLR translation start site. Expression of aflR thus appears to be autoregulated. Increased expression of aflatoxin biosynthetic genes in the transformant might result from an elevated basal level of AFLR, allowing it to overcome nitrate inhibition and to bind to the aflR promotor region, thereby initiating aflatoxin biosynthesis. Results further suggest that aflR is involved in the regulation of multiple parts of the aflatoxin biosynthetic pathway.