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United States Department of Agriculture

Agricultural Research Service

2007 Annual Report

1a.Objectives (from AD-416)
(1) Understand the molecular basis for fungal responses to conditions encountered during invasion of crops in order to identify and modify the factors in corn and other crops that could induce or impede aflatoxin formation or fungal development. Accomplishment of objective 1 could lead to generic approaches to enhance resistance in all crops vulnerable to aflatoxin contamination. (2) Determine, by genetic and physiological studies of diverse aflatoxin producing species, whether aflatoxin production provides an adaptive advantage for fungal survival and invasion of crops, particularly because many natural isolates of A. flavus do not produce aflatoxins. (3) Determine the molecular responses of aflatoxin producing fungi to stress factors, particularly with regard to developing an understanding of the ability of the fungi to adapt and produce toxins. (4) Undertake and utilize newly available sequences of genomic DNA from Aspergillus species to develop rapid and highly sensitive PCR based tests to identify aflatoxigenic fungi and their toxins in contaminated food products.

1b.Approach (from AD-416)
(1) Microarray experiments will be used to identify candidate genes in A. flavus that are turned on or off during a variety of environmental and nutritional conditions that are known to alter gene expression affecting aflatoxin biosynthesis. The DNA microarrays will exploit the available genomic resources of A. flavus ESTs and the whole genome sequences, combined with statistical analysis of up and down regulated signals on the chips. The candidate genes identified from microarrays (both A. flavus EST and whole genome) will be verified or confirmed through RT-Q- PCR or other well established methods. For further analysis, to identify specific genes, targeted gene mutagenesis will be necessary to determine their biological function. Comparison of the gene expression data under these aflatoxin-producing and non-producing conditions will allow us to identify the specific genes expressed under aflatoxin-producing conditions. (2) The adaptive advantage of aflatoxin production in certain environments will be determined by genetic and physiological studies. It will be ascertained if gene cluster imparts some fitness advantage in some environmental niches, particularly when aflatoxin production does not appear to be a virulence factor for crop infestation. Isogenic isolates will be developed in A. flavus for this experiment. Fungal viability will be measured for up to 12 months with these isolates. The measure of longevity proposed in the present study as a measure of fitness will be an important determinant for understanding aflatoxin production in Aspergillus populations in crop soils. Comparisons of genomic sequences will be made between toxigenic A. flavus found in agricultural fields and other Aspergillus species (non-toxigenic and domesticated) to determine what genes are involved in fungal virulence and toxin production of A. flavus, as well as its ability to survive in the field. (3) Stress cues that change the activity of proteins in the biosynthetic pathway and gene transcription will be evaluated. The chromatin structure of the gene cluster and adjacent regions will be studied after exposure to aflatoxin inducing conditions. Parameters such as DNA methylation patterns, that alter chromatin structure, will be explored in these adjacent regions to determine how aflatoxin biosynthesis is turned on in the fungus. Understanding environmental stress cues affecting aflatoxin gene expression may help to develop strategies to reduce aflatoxin contamination of corn under field conditions. It will be determined if the drought tolerant varieties have lower levels of aflatoxin contamination because the host-fungus interaction somehow alters the ability of the fungus to initiate toxin production at the DNA level. (4) The use of PCR to quantify the level of toxigenic fungi in foods through the use of Taqman primer-probe assay will be assessed to provide information on fungal bioburden, but not the level of aflatoxin contamination. The test will be designed to measure the potential of the commodity to become severely contaminated with the fungus (and consequently toxin) if stored under conditions suitable for subsequent growth of the fungus.

3.Progress Report
This ARS research project is also the parent project of the following cooperative agreements involved in like research: Johns Hopkins University, Baltimore, Maryland/6435-41420-005-01S; Xavier University, New Orleans, Louisiana/6435-41420-005-03S; Tohoku University, Tohoku, Japan/6435-41420-005-04M; TIGR/6435-41420-005-05S (expired); National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan/6435-41420-005-06N; and TIGR/6435-41420-005-07G (new).

1. Proof of involvement of genes in aflatoxin synthesis and fungal development obtained. -- It is important that genes involved in aflatoxin formation be identified so that they can be targeted for inhibition. Aflatoxin is a potent fungal toxin produced by Aspergillus flavus and A. parasiticus sometimes contaminating corn and other crops and causing large economic losses. Experiments using microarray technology (slides containing spots of deoxyribonucleic acid [DNA]) have resulted in the identification of several hundred genes that are up- or down-regulated under different environmental conditions that are known to modulate aflatoxin production such as temperature, pH (acidity and alkalinity), and carbon and nitrogen nutrient sources. Further, the genetic basis for loss of aflatoxin production in toxin-deficient mutants of A. parasiticus (generated by physical manipulation of toxin-producing strains) has been investigated using microarrays and metabolic profiling; specific regulatory genes causing this loss have been identified. Microarray experiments have been completed and candidate gene expression profiles have been validated for the genes that are potentially involved in the control of fungal development and secondary metabolism (dependent on the global regulatory gene veA). Additionally, the functions of three genes in the aflatoxin biosynthesis gene cluster were characterized after knocking out the function of these genes by genetic manipulation to decipher their exact role in toxin production. 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.

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.

5.Significant Activities that Support Special Target Populations

6.Technology Transfer

Number of non-peer reviewed presentations and proceedings2

Review Publications
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.

Last Modified: 12/1/2015
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