2007 Annual Report
2. DNA probes (primer sets) identified for universal screening for genetic variability of Aspergillus group fungi. -- Specific DNA-based methods are needed to identify strains of the aflatoxin producing fungus, A. flavus which produces aflatoxin; aflatoxin is a potent fungal toxin sometimes contaminating corn and other crops and causing large economic losses due to disposal of sometimes large quantities of contaminated commodities. Primer sets were developed for specific identification of genetic variability within the aflatoxin biosynthesis gene cluster or in other parts of the fungal genome in aflatoxin-producing and aflatoxin non-producing fungi. With the determination of the variability in the genetic organization in these fungi, a phenomenon called on genetic drift (movement of genetic information) is being examined as the driving force responsible for the loss of the entire aflatoxin gene cluster in non-aflatoxigenic A. flavus isolates, when aflatoxins have lost their adaptive value in nature. We are in the process of preparing knockout constructs to produce an isogenic (genetically similar) series of A. flavus and A. parasiticus aflatoxin pathway defective mutants that we hope will allow us to assess whether or not production of aflatoxin or its precursors contributes to colony survival and the fungus’ ability to adapt to stress conditions as measured by viable spore production and colony formation. Studies on the molecular characterization of the aflatoxin pathway from the aflatoxigenic cousin of A. flavus, namely toxin-producing A. ochraceoroseus, A. rambelli, as well as non-toxigenic A.oryzae, continues to determine if aflatoxin production provides a competitive advantage to A. flavus for its ability to survive in field conditions. This research is covered under the National Food Safety Action Plan (National Program 108). Component 2, Mycotoxins and Plant Toxins, Problem Statement 2.1.2: Crop/Fungal/Insect/Toxin Relationships, Problem Statement 2.1.3: Production Practices and Expert Systems and Problem Statement 2.1.5: Biocontrol Technologies.
3. Role of antioxidant compounds or stress metabolic pathways on toxin biosynthesis and fungal developmental processes determined by microarray and other molecular methods. -- Now that the whole genome sequencing of the aflatoxin producing fungus, A. flavus, has been completed, it has opened up many avenues for defining the role of specific fungal genes in aflatoxin production, particularly the ones that are activated during the exposure of the fungus to stress conditions leading to aflatoxin contamination of the host plant. Aflatoxin is a potent fungal toxin sometimes contaminating corn and other crops and causing large economic losses due to disposal of sometimes large quantities of contaminated commodities. We have successfully used microarray studies to identify many genes that may play a critical role in the fungal response to, for example, antioxidants from walnut shells. In addition, from our recent studies using microarray analysis on defining the expression profile of genes related to aflatoxin biosynthesis in A. flavus, the involvement of antioxidant enzymes has been evidenced. The yeast system of Saccharomyces cerevisiae (name of yeast) has been used as a model organism for understanding the cellular responses to treatment with oxidant and antioxidant compounds, attempting to establish a relationship of these reactions and secondary metabolism. The specific role of the genes of interest is currently being determined using gene knockout techniques. The ultimate goal is to develop breeds of crops with traits that interfere with the processes that induce aflatoxin formation, such as oxidative stress. This research is covered under the National Food Safety Action Plan (National Program 108). Component 2, Mycotoxins, Problem Statement 2.1.4: Breeding resistant crops.
4. Primer sets selected and developed for distinguishing aflatoxigenic from non- aflatoxigenic strains of Aspergillus species. The sequencing of the entire DNA of A. flavus has been completed, and in other labs several other genomes of aflatoxin non-producing Aspergillus species have been studied, for example, A. fumigatus, which is a human pathogen; A. oryzae, which is used in food fermentation; A. niger, which is used in industrial fermentation; and A. nidulans, widely considered as a model fungus for biological studies. Comparisons between the genomes of A. flavus, A. fumigatus, A. oryzae, and A. nidulans have allowed us to identify significant differences at the gene level, particularly in the expression patterns of the gene. For example, in phylogenetic studies (assessment of ancestral relationship between species) we have demonstrated that A. oryzae isolates in one clade (group within a species) strikingly resemble an A. flavus subgroup of non-aflatoxigenic L-type isolates and may descend from certain non-aflatoxigenic L-type A. flavus isolates. Additionally, the distribution of single-nucleotide polymorphisms (SNP = variation caused by a change of a single nucleotide) among A. flavus isolates from well-separated geographic locations in the U.S. showed that genetic recombination among A. flavus isolates from different vegetative compatibility groups does not occur. This information has been very vital in our search for primer sets (short DNA pieces) that will allow us to distinguish one fungal isolate from another and determine the potential of an isolate to contaminate crops with aflatoxin. Therefore, molecular and phylogenetic markers to differentiate strains in the A. flavus group fungi were developed using unique SNP's in (a) the omtA gene which put A. flavus strains collected from various geographic regions into distinct groups, and (b) deletions in the norB-cypA region necessary for the aflatoxin G toxin production which differentiate L and S sclerotia A. flavus strains based upon different size gaps. This research is covered under the National Food Safety Action Plan (National Program 108). Component 2, Mycotoxins and Plant Toxins, Problem Statement 2.1.2: Crop/Fungal/Insect/Toxin Relationships, Problem Statement 2.1.3: Production Practices and Expert Systems and Problem Statement 2.1.5: Biocontrol Technologies.
Duran, R.M., Cary, J.W., Calvo, A.M. 2007. Production of cyclopiazonic acid, aflatrem and aflatoxin by Aspergillus flavus is regulated by veA, a gene necessary for sclerotial formation. Applied Microbiology and Biotechnology. 73(5):1158-1168.
Chang, P.-K., Hua, S.S.T. 2007. Nonaflatoxigenic Aspergillus flavus TX9-8 Competitively Prevents Aflatoxin Accumulation by A. flavus Isolates of Large and Small Sclerotial Morphotypes. International Journal of Food Microbiology. 114:275-279.
Chang, P.-K., Hua, S.T. 2007. Molasses Supplementation Promotes Conidiation but Suppresses Aflatoxin Production by Small Sclerotial Aspergillus flavus. Letters in Applied Microbiology. 44(2):131-137.
Yu, J., Cleveland, T.E. 2007. Aspergillus flavus Genomics for Discovering Genes Involved in Aflatoxin Biosynthesis. In: Rimando, A.M., Baerson, S.R., editors. American Chemical Society Symposium Series: Polyketides - Biosynthesis, Biological Activity, and Genetic Engineering. Washington, DC: American Chemical Society. 955:246-260.
Klich, M.A. 2007. Environmental and developmental factors influencing aflatoxin production by Aspergillus flavus and Aspergillus parasiticus. Mycoscience. 48:71-80.
Klich, M.A. 2006. Identification of clinically relevant Aspergilli. Medical Mycology. 44:5127-5131.
Price, M.S., Yu, J., Nierman, W.C., Kim, H.S., Pritchard, B., Jacobus, C.A., Bhatnagar, D., Cleveland, T.E., Payne, G.A. 2006. The aflatoxin pathway regulator AflR induces gene transcription inside and outside of the aflatoxin biosynthetic cluster. Federation of European Microbiological Societies Microbiology Letters. 255:275-279.
Ehrlich, K.C. 2006. Evolution of the Aflatoxin Gene Cluster. Mycotoxin Research. 22:9-15.
Bhatnagar, D., Cary, J.W., Ehrlich, K., Yu, J., Cleveland, T.E. 2006. Understanding the Genetics of Regulation of Aflatoxin Production and Aspergillus flavus Development. Mycopathologia. 162:155-166.
Cary, J.W., Ehrlich, K. 2006. Aflatoxigenicity in Aspergillus: molecular genetics, phylogenetic relationships and evolutionary implications. Mycopathologia. 162:167-177.
Bhatnagar, D., Proctor, R., Payne, G.A., Wilkinson, J.R., Yu, J., Cleveland, T.E., Nierman, W.C. 2006. Genomics of Mycotoxigenic Fungi, pp. 157-177. In: Barug, D., Bhatnagar, D., van Egmond, H.P., van der Kamp, J.W., van Osenbruggen, W.A. Visconti, A., (eds). The Mycotoxin Factbook. The Netherlands: Wageningen Academic Publishers. 400 p.
Hua, S.T., Tarun, A.S., Pandey, S.N., Chang, L.Y., Chang, P. 2007. Characterization of AFLAV, a Tfl/Sushi retrotransposon from Aspergillus flavus. Mycopathologia. 163(2):97-104.
Ehrlich, K. 2007. Polyketide Biosynthesis in Fungi. In: Rimando, A.M. and Baerson, S.R., editors. Washington, DC: American Chemical Society. American Chemical Society Symposium Series. 955:68-80.