Impact of temperature stress and validamycin A on compatible solutes and fumonisin production in F. verticillioides: role of trehalose-6-phosphate synthase.

Fusarium verticillioides is a pathogen of maize that causes root, stalk and ear rot and produces fumonisins, toxic secondary metabolites associated with disease in livestock and humans. Environmental stresses such as heat and drought influence disease severity and toxin production, but the effects of abiotic stress on compatible solute production by F. verticillioides have not been fully characterized. We found that decreasing the growth temperature leads to a long-term reduction in polyol levels, whereas increasing the temperature leads to a transient increase in polyols. The effects of temperature shifts on trehalose levels are opposite the effects on polyols and more dramatic. Treatment with validamycin A, a trehalose analog with antifungal activity, leads to a rapid reduction in trehalose levels, despite its known role as a trehalase inhibitor. Mutant strains lacking TPS1, which encodes a putative trehalose-6-phosphate synthase, have altered growth characteristics, do not produce detectable amounts of trehalose under any condition tested, and accumulate glycogen at levels significantly higher than wild-type F. verticillioides. TPS1 mutants also produce significantly less fumonisin than wild type and are also less pathogenic than wild type on maize. These data link trehalose biosynthesis, secondary metabolism, and disease, and suggest that trehalose metabolic pathways may be a viable target for the control of Fusarium diseases and fumonisin contamination of maize.

In vitro antioxidant capacity and anti-inflammatory activity of seven common oats.

Oats are gaining increasing scientific and public interest for their purported antioxidant-associated health benefits. Most reported studies focused on specific oat extracts or particular oat components, such as ß-glucans, tocols (vitamin E), or avenanthramides. Studies on whole oats with respect to antioxidant and anti-inflammatory activities are still lacking. Here the antioxidant and anti-inflammatory activities from whole oat groats of seven common varieties were evaluated. All oat varieties had very similar oxygen radical absorption capacity compared with other whole grains. In an anti-inflammatory assay, oat variety CDC Dancer inhibited tumor necrosis factor-α induced nuclear factor-kappa B activation by 27.5% at 2 mg/ml, whereas variety Deiter showed 13.7% inhibition at a comparable dose. Avenanthramide levels did not correlate with the observed antioxidant and anti-inflammatory activities. Further investigations are needed to pinpoint the specific antioxidant and anti-inflammatory compounds, and potential synergistic and/or matrix effects that may help explain the mechanisms of oat's anti-inflammatory actions.

Fusarium verticillioides chitin synthases CHS5 and CHS7 are required for normal growth and pathogenicity.

Fusarium verticillioides is both an endophyte and a pathogen of maize and is a health threat in many areas of the world because it can contaminate maize with fumonisins, a toxic secondary metabolite. We identified eight putative chitin synthase (CHS) genes in F. verticillioides genomic sequence, and phylogenetic evidence shows that they group into seven established CHS gene classes. We targeted two CHSs (CHS5 and CHS7) for deletion analysis and found that both are required for normal hyphal growth and maximal disease of maize seedlings and ears. CHS5 and CHS7 encode a putative class V and class VII fungal chitin synthase, respectively; they are located adjacent to each other and are divergently transcribed. Fluorescent microscopy found that both CHS deficient strains produce balloon-shaped hyphae, while growth assays indicated that they were more sensitive to cell wall stressing compounds (e.g., the antifungal compound Nikkomycin Z) than wild type. Pathogenicity assays on maize seedlings and ears indicated that both strains were significantly reduced in their ability to cause disease. Our results demonstrate that both CHS5 and CHS7 are necessary for proper hyphal growth and pathogenicity of F. verticillioides on maize.

Determination of deoxynivalenol in infant cereal by immunoaffinity column cleanup and high-pressure liquid chromatography-UV detection.

The presence of deoxynivalenol (DON) in cereal-based baby food, a primary source of the first solid food for infants, was studied in order to develop a method to detect its presence at low concentrations. DON, produced primarily by Fusarium graminearum, is commonly isolated from grains and feed around the world and affects both animal and human health, producing diarrhea, vomiting, gastrointestinal inflammation, and immunomodulation. An aqueous extract of infant cereal was cleaned by means of an immunoaffinity chromatography column. After the eluate was evaporated and redissolved, DON was determined by high-pressure liquid chromatography-UV. The level of quantification for DON was 10 ppb for three types of infant cereal (mixed, barley, and oatmeal); the level of detection was 5 ppb. The protocol we have developed can measure DON between 10 to 500 ppb. An advisory level of 1 ppm for wheat products has been established by the U.S. Food and Drug Administration; however, the European Communities (EC) regulations have been set at 200 ppb for cereal-based foods for infants. Only 1 of 52 samples of barley-, mixed-, or oat-based infant cereal purchased in 2008 and 2009 in the United States exceeded the European standard.

Functionalized C-glycoside ketohydrazones: carbohydrate derivatives that retain the ring integrity of the terminal reducing sugar.

Glycosylation often mediates important biological processes through the interaction of carbohydrates with complementary proteins. Most chemical tools for the functional analysis of glycans are highly dependent upon various linkage chemistries that involve the reducing terminus of carbohydrates. However, because of ring opening, the structural integrity of the reducing sugar ring (pyranose or furanose) is lost during these techniques, resulting in derivatized carboydrates that markedly differ from the parent molecule. This paper describes a new aqueous-based, one-pot strategy that involves first converting the sugar to a C-glycoside ketone, followed by conversion to ketohydrazones or oximes. Hence, the C-glycoside ketones are tagged with fluorescence, colored, cationic or biotin-labeled groups or immobilized onto hydrazine-functionalized beads. No activating or protecting groups are required, and the chemistry is mild enough for a wide range of carbohydrates. We demonstrate the versatility of the approach to diverse glycans, including bead immobilization and lectin analysis of acarbose, an antidiabetic drug, to dabsyl-tagged enzyme substrates to screen cellulases, and for the analysis of plant cell wall hemicellulosics.

Opportunities for biotechnology and policy regarding mycotoxin issues in international trade.

Despite being introduced more than a decade ago, agricultural biotechnology still remains framed in controversy impacting both the global economy and international regulations. Controversies surrounding agricultural biotechnology produced crops and foods commonly focus on human and environmental safety, intellectual property rights, consumer choice, ethics, food security, poverty reduction and environmental conservation. Originally, some consumers were reluctant to accept the first generation agricultural biotechnology products because they appeared to primarily benefit agricultural producers; however, it is clear from continued evaluations that these technologies also improved both the safety and wholesomeness of food and helped improve the environment. Plants engineered to resist insect pests and tolerate less toxic pesticides resulted in improved yields thereby enabling farmers to produce more food per acre while reducing the need for herbicides, pesticides, and water and tilling. An indirect benefit of reduced pest damage in transgenic corn expressing genes to control insect pests is lower levels of mycotoxins, most notably those caused by the genus Fusarium. Mycotoxins are an important regulatory issue globally because of their toxic and carcinogenic potential to humans and animals. Complicating this issue is the fact that toxicological databases for mycotoxins are relatively incomplete compared to other food contaminants. Current debates about agricultural biotechnology and mycotoxins reveal significant differences in perception of associated risks and benefits. When faced with uncertainty, regulators tend to set limits as low as possible. Additionally, some regulators invoke the "Precautionary Principle" when limited information is available or disputes over interpretation exist for possible contaminants, including mycotoxins. A major concern regarding use of the "Precautionary Principle" is the appearance that regulators can justify setting any limit on the basis of inconclusive or unknown potential hazards of a contaminant which may significantly impact global trade because mycotoxin residues vary widely between countries. This paper describes the current economic and heath impact of these regulations and their impact on international trade.

Real-time PCR assay to quantify Fusarium graminearum wild-type and recombinant mutant DNA in plant material.

Fusarium graminearum (teleomorph, Gibberella zeae) is the predominant causal agent of Fusarium head blight (FHB) of wheat resulting in yearly losses through reduction in grain yield and quality and accumulation of fungal generated toxins in grain. Numerous fungal genes potentially involved in virulence have been identified and studies with deletion mutants to ascertain their role are in progress. Although wheat field trials with wild-type and mutant strains are critical to understand the role these genes may play in the disease process, the interpretation of field trial data is complicated by FHB generated by indigenous species of F. graminearum. This report describes the development of a SYBR green-based real time PCR assay that quantifies the total F. graminearum genomic DNA in a plant sample as well as the total F. graminearum genomic DNA contributed from a strain containing a common fungal selectable marker used to create deletion mutants. We found our method more sensitive, reproducible and accurate than other similar recently described assays and comparable to the more expensive probe-based assays. This assay will allow investigators to correlate the amount of disease observed in wheat field trials to the F. graminearum mutant strains being examined.

Fusarium graminearum TRI14 is required for high virulence and DON production on wheat but not for DON synthesis in vitro.

Fusarium head blight (FHB) of wheat (Triticum aestivum L.), caused by the fungus Fusarium graminearum, is a major concern worldwide. FHB grain is reduced in yield, may fail to germinate, and is often contaminated with deoxynivalenol, a trichothecene mycotoxin linked to a variety of animal diseases and feed refusals. Annual losses in the tens of millions of dollars due to FHB underscore the need to develop improved methods of disease control and prevention. Previous research has identified deoxynivalenol biosynthesis as a virulence factor on wheat. Recently, we found that the TRI14 gene of F. sporotrichioides, closely related to F. graminearum, was not required for synthesis of a related trichothecene, T-2 toxin. TRI14 does not share similarity with any previously described genes in the databases. In this study, we examined the role that F. graminearum TRI14 may play in both deoxynivalenol synthesis and in virulence on wheat. TRI14 deletion mutants synthesize deoxynivalenol on cracked maize kernel medium and exhibit wild-type colony morphology and growth rate on complex and minimal agar media. However, FHB assays on greenhouse-grown wheat indicate that FgDeltaTri14 mutants cause 50-80% less disease than wild type and do not produce a detectable quantity of deoxynivalenol on plants. We discuss a number of possible roles that TRI14 may play in the disease process.

Comparative analysis of 87,000 expressed sequence tags from the fumonisin-producing fungus Fusarium verticillioides.

Fusarium verticillioides (teleomorph Gibberella moniliformis) is a pathogen of maize worldwide and produces fumonisins, a family of mycotoxins that have been associated with several animal diseases as well as cancer in humans. In this study, we sought to identify fungal genes that affect fumonisin production and/or the plant-fungal interaction. We generated over 87,000 expressed sequence tags from nine different cDNA libraries that correspond to 11,119 unique sequences and are estimated to represent 80% of the genomic complement of genes. A comparative analysis of the libraries showed that all 15 genes in the fumonisin gene cluster were differentially expressed. In addition, nine candidate fumonisin regulatory genes and a number of genes that may play a role in plant-fungal interaction were identified. Analysis of over 700 FUM gene transcripts from five different libraries provided evidence for transcripts with unspliced introns and spliced introns with alternative 3' splice sites. The abundance of the alternative splice forms and the frequency with which they were found for genes involved in the biosynthesis of a single family of metabolites as well as their differential expression suggest they may have a biological function. Finally, analysis of an EST that aligns to genomic sequence between FUM12 and FUM13 provided evidence for a previously unidentified gene (FUM20) in the FUM gene cluster.

Functional demarcation of the Fusarium core trichothecene gene cluster.

Many Fusarium species produce toxic sesquiterpenoids known as trichothecenes, including deoxynivalenol and nivalenol by Fusarium graminearum and T-2 toxin by Fusarium sporotrichioides. These toxins are potent inhibitors of protein synthesis and are a significant agricultural problem due to their adverse affect on human, animal, and plant health. Previously, 10-12 co-regulated orthologous genes within a 26-kb region were identified in F. graminearum and F. sporotrichioides, respectively. A majority of these clustered genes have been shown to be involved in different aspects of trichothecene metabolism including 7 of 15 biosynthetic steps. Three other biosynthetic steps are carried out by genes located elsewhere in the genome. In this study, we sequenced 14-16 kb of DNA on both sides of the core clusters and identified 12 new ORFs in both Fusarium species. Although the predicted functions of some of the new ORFs are consistent with some unassigned biochemical reactions, gene expression and gene deletion studies indicate that none are required for trichothecene biosynthesis. These results provide evidence to demarcate both ends of the core trichothecene gene cluster. Index descriptors: Fungal secondary metabolite, Pathogenic fungi, Gene cluster, Fusarium, Trichothecene, DON

Liquid chromatographic determination of fumonisins B1, B2, and B3 in corn silage.

Corn silage was dried, ground, and then extracted with 0.1 M ethylenediaminetetraacetic acid. The filtrate was applied to a FumoniTest immunoaffinity column. Fumonisins were derivatized with naphthalene-2,3-dicarboxaldehyde, separated on a C(18) liquid chromatographic column, and detected by fluorescence. The detection limits for fumonisin B(1), fumonisin B(2), and fumonisin B(3) were 50, 25, and 25 ng/g of dried silage, respectively. Recoveries of fumonisin B(1), fumonisin B(2), and fumonisin B(3) from wet and dried corn silage spiked over the range of 100-5000 ng/g averaged 91-106%. The method was applied to corn silage samples collected from the midwestern area of the United States during 2001-2002. Of 89 corn silage samples, fumonisin B(1), fumonisin B(2), and fumonisin B(3) were found in 86 (97%), 64 (72%), and 51 (57%) of the samples. The mean positive levels of fumonisin B(1), fumonisin B(2), and fumonisin B(3) were 615, 93, and 51 ng/g, respectively, in dried silage. This suggests that fumonisins may be frequent low level contaminants in corn silage.