2008 Annual Report 1a.Objectives (from AD-416) Identify genes that regulate fumonisin production in F. verticillioides. Identify genes that contribute to the ability of F. verticillioides to cause maize ear rot. Identify biochemical changes in maize in response to fumonisin B1 and F. verticillioides, and to determine if there is cross-talk between maize and F. verticillioides sphingolipids. Identify genes responsible for the biosynthesis of other agronomically important mycotoxins produced by the maize ear rot pathogens F. verticillioides, F. proliferatum and F. subglutinans. Develop a rapid, selective and robust PCR-based diagnostic test to detect and quantify mycotoxigenic strains of Fusarium in contaminated grain and food products. 1b.Approach (from AD-416) Utilize a combination of molecular genetic approaches such as genomic resources and microarray analysis to identify and characterize. 1)genes involved in the regulation of fumonisin production in the fungus Fusarium verticillioides and. 2)genes that contribute to the ability of the fungus to cause maize diseases. Employ chemical analyses (e.g. mass spectroscopy) to determine whether fumonisins affect sphingolipid metabolism in maize. Use molecular genetic methods to identify genes required for the biosynthesis of other mycotoxins produced by agronomically important Fusarium species. Use polymerase chain reaction (PCR) approaches to detect and quantify mycotoxin-producing fungi in maize plants. 3.Progress Report Fusarium verticillioides and some closely related fungal species can cause ear and stalk rot of maize (corn) and produce mycotoxins, including the carcinogenic fumonisins, in infected kernels. Knowledge of the genes that regulate fumonisin production in Fusarium species or that enable the genes to cause disease could contribute to development of control strategies. We are using microarray analysis to examine expression of genes in mutant strains of F. verticillioides that are affected in the regulation of fumonisin production. In the mutants, expression of the fumonisin biosynthetic gene cluster is blocked. In order to identify targets to control maize ear rot caused by F. verticillioides, we are using microarray analysis to identify F. verticillioides genes that are expressed during infection of kernels of developing ears of the model maize line B73 and in developed kernels of a maize line with increased resistance to the fungus. We continue to examine alternate splicing of messenger ribonucleic acid (mRNA) in F. verticillioides. Alternate splicing (AS) involves the differential excision of intron sequences from mRNA molecules. Over 6.3% of F. verticillioides genes, including almost half of those located in the fumonisin biosynthetic gene cluster, undergo AS. In other organisms, AS is an important mechanism of gene regulation. Thus, AS could affect regulation of mycotoxin production in Fusarium. F. proliferatum produces fumonisin mycotoxins and causes multiple crop diseases, including black point of wheat. A survey of naturally infected wheat exhibiting black point revealed that some grain samples contained low levels of fumonisins and F. proliferatum. Inoculation of F. proliferatum strains into wheat resulted in black point symptoms and contamination of grain with fumonisins. We have also developed a genetic transformation system to disrupt F. proliferatum genes. Efforts to disrupt the fumonisin biosynthetic gene FUM1 are in progress. We are examining whether there is a relationship between resistance to ear rot and insensitivity to fumonisins by conducting field experiments with maize lines that are highly insensitive to the mycotoxin fumonisin B1. The fumonisin B1-insensitive lines are being inoculated with fumonisin-producing and nonproducing strains of F. verticillioides to assess their relative resistance to ear rot. Multiple Fusarium metabolites, including some mycotoxins, are polyketides. Knowledge of the structure of polyketide synthase (PKS) genes should lead to identification of conserved physiological processes critical to fungal growth and disease. Phylogenetic analysis of the 58 PKS genes from the genome sequences of four Fusarium species identified three well conserved sets of PKSs. These four Fusarium species have three PKS genes in common, while F. oxysporum and F. verticillioides have nine genes in common. Despite overlapping ecological nitches of F. verticillioides and F. graminearum, both occur on maize, the two species have only five PKS genes in common. This research was performed under NP 108, Component 2. 4.Accomplishments 1. NATURAL OCCURRANCE OF FUMONISINS IN WHEAT. Fumonisin mycotoxins can accumulate in maize (corn) kernels and are of concern because they can cause several animal diseases, including cancer and neural tube defects in laboratory rodents. The toxins are also epidemiologically associated with esophageal cancer and neural tube defects in some human populations for which maize is a dietary staple. Fumonisins are produced by several Fusarium species, including Fusarium proliferatum, a pathogen of maize, wheat and several other crop species. On wheat, F. proliferatum can cause black point disease of grain. A survey of multiple samples of field-grown wheat with black point symptoms revealed the presence of low levels of fumonisins in some grain samples. Fumonisin-producing Fusarium species F. proliferatum, F. fujikuroi, and F. nygamai were isolated from some of the wheat samples. When some of the F. proliferatum strains were inoculated into greenhouse-grown wheat, the inoculated wheat exhibited black point symptoms and low levels of fumonisin contamination. The results indicate that field grown wheat that is naturally infected with Fusarium as well as green-house grown wheat that has been artificially inoculated with F. proliferatum can contain fumonisins. However, the levels of fumonisins that accumulated were low relative to levels that can accumulate in maize. This accomplishment addresses NP 108, Component 2, Problem Area 2.1.4 and 2.1.2. 5.Significant Activities that Support Special Target Populations None. 6.Technology Transfer Number of New Commercial Licenses Executed 1 Number of Non-Peer Reviewed Presentations and Proceedings 1 Review Publications Bluhm, B., Kim, H., Butchko, R.A., Woloshuk, C.P. 2008. Involvement of ZFR1 of Fusarium verticilliodes in kernel colonization and the regulation of FST1, a putative sugar transporter gene required for fumonisin biosynthesis on maize kernels. Molecular Plant Pathology. 9(2):203-211. Desjardins, A.E., Proctor, R. 2007. Molecular biology of Fusarium mycotoxins. International Journal of Food Microbiology. 119(1-2):47-50. Desjardins, A.E., Busman, M., Proctor, R., Stessman, R.J. 2007. Wheat kernel black point and fumonisin contamination by Fusarium proliferatum. Journal of Food Additives & Contaminants. 24(10):1131-1137. Desjardins, A.E., Busman, M., Muhitch, M.J., Proctor, R. 2007. Complementary host-pathogen genetic analyses of the role of fumonisins in the Zea mays-Gibberella moniliformis seedling interaction. Physiological and Molecular Plant Pathology. 70(4-6):149-160. Proctor, R., Busman, M., Seo, J., Lee, Y., Plattner, R.D. 2008. A fumonisin biosynthetic gene cluster in Fusarium oxysporum strain O-1890 and the genetic basis of B versus C fumonisin production. Fungal Genetics and Biology. 45:1016-1026. Available: www.elsevier.com/locate/yfgbi. Proctor, R., Butchko, R.A., Brown, D.W., Moretti, A. 2007. Functional characterization, sequence comparisons and distribution of a polyketide synthase gene required for perithecial pigmentation in some Fusarium species. Journal of Food Additives & Contaminants. 24(10):1076-1087.