Bruce C. Campbell

Activities of conventional antifungal agents, fludioxonil, strobilurin and antimycin A, which target the oxidative and osmotic stress response system

The activity of fludioxonil, a phenylpyrrole fungicide, is elevated by coapplication of the aspirin/salicylic acid metabolite, 2,5-dihydroxybenzoic ac

Aims: Identify natural products that effectively target antioxidative signal transduction/stress response systems [i.e., mitogen‐activated protein kinase (MAPK) pathway, mitochondrial superoxide dismutase (Mn‐SOD)] of fungi. Enhance activity of strobilurin or fludioxonil with discovered compounds. Methods and Results: Enhancement of antifungal activity of strobilurins, inhibitors of complex III of the mitochondrial respiratory chain, was tested using berberine hemisulfate and different phenolic compounds. The Saccharomyces cerevisiae sod2Δ, a deletion mutant lacking Mn‐SOD gene, was highly sensitive to berberine and veratraldehyde. Functional complementation analysis verified these compounds target Mn‐SOD. Activity of strobilurin (25–50 μmol l^‐1) was elevated on most aspergilli and Penicillium expansum by co‐application with berberine or veratraldehyde (2–4 mmol l^‐1). These compounds also prevented Aspergillus fumigatus MAPK mutants (sakAΔ and mpkCΔ) from escaping toxicity of fludioxonil (a phenylpyrrole fungicide potentiated by the MAPK pathway), a typical phenotype of fungal MAPK mutants. Conclusions: Strobilurin activity or prevention of fungal escape from fludioxonil toxicity can be enhanced by co‐application of certain alkaloids or phenolics. Significance and Impact of the Study: Natural products can be used to target cellular stress response systems in fungal pathogens and serve as alternatives/additives to commercial antifungal agents.

Aims: The aim of this study was to show whether antioxidative response systems are potentially useful molecular targets for control of Aspergillus fumigatus and Aspergillus flavus. Selected phenolic agents are used in target‐gene‐based bioassays to determine their impact on mitochondrial respiration. Methods and Results: Vanillyl acetone, vanillic acid, vanillin, cinnamic acid, veratraldehyde, m‐coumaric acid (phenolic agents to which Saccharomyces cerevisiae sod2Δ mutant showed sensitivity), carboxin (inhibits complex II of the mitochondrial respiratory chain), strobilurins/antimycin A (inhibits complex III of the mitochondrial respiratory chain) and fludioxonil/fenpiclonil [antifungals potentiated by mitogen‐activated protein kinase (MAPK)] were examined in A. fumigatus, A. flavus and S. cerevisiae. Individual or combined application of phenolics with inhibitors of mitochondrial respiration showed some of the phenolics effectively inhibited fungal growth. Target‐gene bioassays were performed using a sakAΔ (MAPK deletion) strain of A. fumigatus and a complementation analysis using the mitochondrial superoxide dismutase (Mn‐SOD) gene (sodA) of A. flavus in the ortholog mutant, sod2Δ, of S. cerevisiae. The results demonstrated that mitochondrial antioxidative stress system plays important roles in fungal response to antifungal agents tested. Conclusions: Antioxidative response systems of fungi can be an efficient molecular target of phenolics for pathogen control. Combined application of phenolics with inhibitors of mitochondrial respiration can effectively suppress the growth of fungi. Significance and Impact of the Study: Natural compounds that do not pose any significant medical or environmental risks could serve as useful alternatives or additives to conventional antifungals. Identifying the antioxidative response systems in other pathogens could improve methods for fungal control.

It was initially shown that gallic acid, from hydrolysable tannins in the pelliele of walnut kernels, dramatically inhibits biosynthesis of aflatoxin byAspergillus flavus. The mechanism of this inhibition was found to take place upstream from the gene cluster, including the regulatory gene,aflR, involved in aflatoxin biosynthesis. Additional research using other antioxidant phenolics showed similar antiaflatoxigenic activity to gallic acid. Treatment ofA. flavus withtert-butyl hydroperoxide resulted in an almost doubling of aflatoxin biosynthesis compared to untreated samples. Thus, antioxidative response systems are potentially useful molecular targets for control ofA. flavus. A high throughput screening system was developed using yeast,Saccharomyces cerevisiae, as a model fungus. This screening provided an avenue to quickly identify fungal genes that were vulnerable to treatment by phenolic compounds. The assay also provided a means to quickly assess effects of combinations of phenolics and certain fungicides affecting mitochondrial respiration. For example, theS. cerevisiae sod2† mutant was highly sensitive to treatment by certain phenolics and strobilurins/antimycin A, fungicides which inhibit complex III of the mitochondrial respiratory chain. Verification of stress to this system in the target fungus,A. flavus, was shown through complementation analysis, wherein the mitochondrial superoxide dismutase (Mn-SOD) gene (sodA) ofA. flavus in the ortholog mutant,sod2†, ofS. cerevisiae, relieved phenolic-induced stress. Mitochondrial antioxidative stress systems play an important role in fungal response to antifungals. Combined treatment of fungi with phenolics and inhibitors of mitochondrial respiration can effectively suppress growth ofA. flavus in a synergistic fashion.

The antioxidative stress-response system is essential to fungi for tolerating exposure to phenolic compounds. We show how this system can be targeted to improve fungal control by using compounds that inhibit the fungal mitochondrial respiratory chain. Targeting mitochondrial superoxide dismutase with selected phenolic acid derivatives (e.g., vanillyl acetone) resulted in a 100- to 1,000-fold greater sensitivity to strobilurin or carboxin fungicides. This synergism is significantly greater with strobilurin than with carboxin, suggesting that complex III of the mitochondrial respiratory chain is a better target than complex II for fungal control, using phenolics. These results show certain natural compounds are effective synergists to commercial fungicides and can be used for improving control of food-contaminating pathogens. These results suggest that the use of such compounds for fungal control can reduce environmental and health risks associated with commercial fungicides, lower cost for control, and the probability for development of resistance.

Saccharomyces cerevisiae served as a model fungal system to examine functional genomics of oxidative stress responses and reactions to test antioxidant compounds. Twenty-two strains of S. cerevisiae, including a broad spectrum of singular gene deletion mutants, were exposed to hydrogen peroxide (H₂O₂) to examine phenotypic response to oxidative stress. Responses of particular mutants treated with gallic, tannic or caffeic acids, or methyl gallate, during H₂O₂ exposure, indicated that these compounds alleviated oxidative stress. These compounds are also potent inhibitors of aflatoxin biosynthesis in Aspergillus flavus. To gain further insights into a potential link between oxidative stress and aflatoxin biosynthesis, 43 orthologs of S. cerevisiae genes involved in gene regulation, signal transduction (e.g., SHO1, HOG1, etc.) and antioxidation (e.g., CTT1, CTA1, etc.) were identified in an A. flavus expressed sequence tag library. A successful exemplary functional complementation of an antioxidative stress gene from A. flavus, mitochondrial superoxide dismutase (sodA), in a sod2Δ yeast mutant further supported the potential of S. cerevisiae deletion mutants to serve as a model system to study A. flavus. Use of this system to further examine functional genomics of oxidative stress in aflatoxigenesis and reduction of aflatoxin biosynthesis by antioxidants is discussed.

Larvae of the navel orangeworm (NOW), Amyelois transitella (Walker), a major pest of almonds and pistachios, and the codling moth (CM), Cydia pomonella (L.), the principal pest of walnuts and pome fruits, are commonly found in tree nut kernels that can be contaminated with aflatoxin, a potent carcinogen. The ability of larvae of these insects to metabolize aflatoxin B₁ (AFB1) was examined. A field strain of NOW produced three AFB1 biotransformation products, chiefly aflatoxicol (AFL), and minor amounts of aflatoxin B_2a (AFB2a) and aflatoxin M₁ (AFM1). With AFL as a substrate, NOW larvae produced AFB1 and aflatoxicol M₁ (AFLM1). A lab strain of CM larvae produced no detectable levels of AFB1 biotransformation products in comparison to a field strain which produced trace amounts of only AFL. Neither NOW nor CM produced AFB₁-8,9-epoxide (AFBO), the principal carcinogenic metabolite of AFB1. In comparison, metabolism of AFB1 by chicken liver yielded mainly AFL, whereas mouse liver produced mostly AFM1 at a rate eightfold greater than AFL. Mouse liver also produced AFBO. The relatively high production of AFL by NOW compared to CM may reflect an adaptation to detoxify AFB1. NOW larvae frequently inhabit environments highly contaminated with fungi and, hence, aflatoxin. Only low amounts, if any, of this mycotoxin occur in the chief CM hosts, walnuts, and pome fruits. Characterizations of enzymes and co-factors involved in biotransformation of AFB1 are discussed. Arch. Insect Biochem. Physiol. 45:166–174, 2000. Published 2001 Wiley-Liss, Inc.