Effect of Carotenoids on Aflatoxin B(1) Synthesis by Aspergillus flavus

ABSTRACT Carotenes and xanthophylls occurring in yellow corn and related terpenoids were tested for their effect on growth and aflatoxin B(1) production by Aspergillus flavus NRRL 3357, using the suspended disc culture method. Aflatoxin synthesis was inhibited at concentrations of beta-carotene, lutein, and zeaxanthin comparable to those found in the horny endosperm of mature corn. Usually growth was not significantly affected. Inhibition of aflatoxin biosynthesis was greater for compounds with an alpha-ionone-type ring (alpha-carotene, lutein, or alpha-ionone) compared with compounds with a beta-ionone ring. The presence of hydroxy groups on the rings tended to decrease inhibition, but did not override the effect of the ring type; lutein was similar to alpha-carotene and zeaxanthin was similar to beta-carotene in inhibition. A mutant accumulating norsolorinic acid (NA), A. parasiticus SRRC 162, incubated with alpha-carotene produced reduced levels of both NA and aflatoxin, indicating that inhibition occurred before NA. Additional A. flavus strains tested against 50 mug/ml of beta-carotene had 89 to 96% inhibition, which was significantly more sensitive than NRRL 3357. A. parasiticus strains were less sensitive and generally had similar or lower inhibition than NRRL 3357. The results indicate that the presence of carotenoids in endosperm may decrease the amount of aflatoxin produced by A. flavus.     

Advances in the Development of Host Resistance in Corn to Aflatoxin Contamination by Aspergillus flavus

ABSTRACT Aflatoxins are toxic, highly carcinogenic secondary metabolites of Aspergillus flavus and A. parasiticus, which when produced during fungal infection of a susceptible crop in the field or after harvest contaminate food and feed and threaten human and animal health. Although there are several management strategies that may reduce aflatoxin contamination of corn, the preeminent strategy for elimination of aflatoxin is to develop preharvest host resistance to aflatoxin accumulation. This strategy has gained even greater prominence due to recent discoveries of natural resistance in corn that can be exploited in plant-breeding strategies. The ability to identify resistant corn genotypes has been enhanced by the development of a laboratory kernel-screening assay and by a strain of A. flavus genetically engineered to produce beta-glucuronidase, an enzyme whose activity can be monitored to assess the degree of fungal infection in kernels. Investigations of resistant corn genotypes have associated kernel pericarp wax characteristics with resistance, identified kernel proteins associated with resistance to and inhibition of fungal growth or aflatoxin biosynthesis, and identified chromosome regions associated with resistance to Aspergillus ear rot and aflatoxin production. Such research advances could lead, in the near future, to commercially available, agronomically acceptable corn lines with multiple preharvest resistances to aflatoxin contamination.     

Germination induces accumulation of specific proteins and antifungal activities in corn kernels

ABSTRACT This study examined protein induction and accumulation during imbibition and germination of corn kernels, as well as antifungal activities of extracts from germinating kernels against Aspergillus flavus and Fusarium moniliforme. Genotypes studied included GT-MAS:gk and Mp420, which are resistant to A. flavus infection and aflatoxin accumulation, and Pioneer 3154 and Deltapine G-4666, which are susceptible to A. flavus infection and aflatoxin accumulation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis resolved five protein bands that were present at higher concentrations in germinated kernels than in nongerminated kernels. Western blot analyses revealed that one of these proteins reacted with the 22-kDa zeamatin antiserum, and a zeamatin-like protein accumulated to a higher concentration in germinated kernels. Two protein bands from dry kernels that reacted with ribosome-inactivating protein (RIP) antiserum were identified as the 32-kDa proRIP-like form and an 18-kDa peptide of the two peptides that form active RIP. However, in germinated kernels, two protein bands that reacted with RIP antiserum were identified as two RIP-like peptides with a molecular mass of approximately 18 and 9 kDa. Purified RIP and zeamatin from corn inhibited growth of A. flavus. Bioassays of germinated kernel extracts from all four genotypes exhibited antifungal activity against A. flavus and F. moniliforme, with extracts from the susceptible genotypes showing greater inhibition zones. This study provides evidence of protein induction in corn kernels during imbibition or the early stages of germination, and the induced proteins may be related to our previous findings of germination-associated resistance in the corn kernel, especially in the susceptible kernels.     

A Chitinase from Tex6 Maize Kernels Inhibits Growth of Aspergillus flavus

ABSTRACT The maize inbred Tex6 has resistance to colonization and aflatoxin accumulation by Aspergillus flavus. A protein inhibitory to growth of A. flavus has been identified from aqueous extracts of mature Tex6 seeds. This study reports the purification of a chitinase associated with this inhibitory activity to electrophoretic homogeneity and the further characterization of its properties. The inhibitory protein, which has an M(r) of 29,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is an endochitinase that is also capable of exochitinase activity. The enzyme has an optimal pH of 5.5 and a temperature optimum of 45 degrees C. Chitinase activity in maize kernels peaked approximately 36 days after pollination. The Tex6 chitinase purified in this study is capable of inhibiting the growth of A. flavus by 50% at a concentration of 20 mug/ml. Our data indicate that chitinase activity in Tex6 kernels makes a major contribution to the antifungal activity in this maize genotype. Partial peptide sequence of the chitinase showed it to differ from previously reported chitinases.     

A Corn Trypsin Inhibitor with Antifungal Activity Inhibits Aspergillus flavus alpha-Amylase

ABSTRACT In this study, we found that the inhibition of fungal growth in potato dextrose broth (PDB) medium by the 14-kDa corn trypsin inhibitor (TI) protein, previously found to be associated with host resistance to aflatoxin production and active against various fungi, was relieved when exogenous alpha-amylase was added along with TI. No inhibitory effect of TI on fungal growth was observed when Aspergillus flavus was grown on a medium containing either 5% glucose or 1% gelatin as a carbon source. Further investigation found that TI not only inhibited fungal production of extracellular alpha-amylase when A. flavus was grown in PDB medium containing TI at 100 mug ml(-1) but also reduced the enzymatic activity of A. flavus alpha-amylase by 27%. At a higher concentration, however, TI stimulated the production of alpha-amylase. The effect of TI on the production of amyloglucosidase, another enzyme involved in starch metabolism by the fungus, was quite different. It stimulated the production of this enzyme during the first 10 h at all concentrations studied. These studies suggest that the resistance of certain corn genotypes to A. flavus infection may be partially due to the ability of TI to reduce the production of extracellular fungal alpha-amylase and its activity, thereby limiting the availability of simple sugars for fungal growth. However, further investigation of the relationship between TI levels and fungal alpha-amylase expression in vivo is needed.     

Inhibition of mycelial growth and sporidial formation in the wheat pathogenNeovossia indica by a polyamine biosynthetic inhibitor

DL-α-Difluoromethylornithine (DFMO), a specific suicide inhibitor of the polyamine biosynthetic enzyme ornithine decarboxylase (EC 4.1.1.17), strongly inhibited mycelial growth and sporidial formation of the wheat pathogen,Neovossia indica, in vitro, while DL-α-difluoromethylarginine (DFMA), the analogous suicide inhibitor of arginine decarboxylase (EC 4.1.1.19), did not. The inhibited mycelial growth and sporidial formation were not only restored by putrescine (polyamine) addition, but were actually enhanced in the putrescine + DFMO cultures. Besides altering mycelial growth and morphology, DFMO also reduced the cell size drastically. The inhibition of fungal polyamine biosynthesis is discussed in relation to selective control of plant disease.     

Inheritance of resistance to Aspergillus ear rot and aflatoxin production of corn from tex6

ABSTRACT The inheritance of resistance to Aspergillus ear rot and aflatoxin production in corn (Zea mays L.) caused by Aspergillus flavus was studied in progeny derived from crosses between the resistant corn inbred cv. Tex6 and susceptible inbred cvs. B73 and Mo17. From 1994 to 1996, plant generations included were the P(1) (susceptible B73 or Mo17), P(2) (resistant Tex6), F(1), F(2), F(3), BCP(1), BCP(1)-selfed, and BCP(2). The BCP(2)-selfed generation was added in 1995 and 1996 for the B73 x Tex6 cross. Primary ears were pinboard inoculated and evaluated for Aspergillus ear rot severity. F(1) means deviated from the midparent value toward resistance for aflatoxin production and toward susceptibility for ear rot in both crosses. Analyses of generation means indicate that additive gene action was most important in the resistance to both ear rot and aflatoxin production in the B73 x Tex6 cross. Mo17 was somewhat resistant to both traits, so resistance from Tex6 was not well defined in this cross. Broad-sense heritabilities for ear rot and aflatoxin production were 58 and 63% for Mo17 x Tex6, and 66 and 65% for B73 x Tex6. Narrow-sense heritabilities for ear rot and aflatoxin production were 39 and 45% for B73 x Tex6. It is estimated that one cycle of selection for resistance within B73 x Tex6 F(3) families would reduce the percentage of ear rot severity by 8.5% and aflatoxin concentration by 19 ng/g.     

Resistance to Aspergillus flavus in Corn Kernels Is Associated with a 14-kDa Protein

ABSTRACT Corn genotypes resistant or susceptible to Aspergillus flavus were extracted for protein analysis using a pH 2.8 buffer. The profile of protein extracts revealed that a 14-kDa protein is present in relatively high concentration in kernels of seven resistant corn genotypes, but is absent or present only in low concentration in kernels of six susceptible ones. The N-terminal sequence of this 14-kDa protein showed 100% homology to a corn trypsin inhibitor. The 14-kDa protein purified from resistant varieties also demonstrated in vitro inhibition of both trypsin activity and the growth of A. flavus. This is the first demonstration of antifungal activity of a corn 14-kDa trypsin inhibitor protein. The expression of this protein among tested genotypes may be related to their difference in resistance to A. flavus infection and subsequent aflatoxin contamination.     

Advances in the potentiometric determination of pka and log kow of pesticides

The phenylpyrrole fungicide fenpiclonil inhibited the metabolism of glucose in mycelium of Fusarium sulphureum Schlecht at a concentration which only slightly inhibited mycelial growth (EC₁₅). At the same concentration, fenpiclonil also inhibited accumulation and, to a greater extent, phosphorylation of 2-deoxy[U-¹⁴C]glucose in starved mycelium loaded with unlabelled 2-deoxyglucose. Fenpiclonil did not affect cell-free phosphorylation of 2-deoxyglucose or the ATP content of mycelium. Therefore, the primary mode of action of the fungicide may be based on inhibition of transport-associated phosphorylation of glucose. This may cause a cascade of metabolic events which eventually lead to fungal growth inhibition and death. One major event affected by inhibition of transport-associated phosphorylation is the accumulation of polyols, such as glycerol and mannitol, in mycelium. This was not observed in an osmotically sensitive, fenpiclonil-resistant laboratory isolate of the fungus.     

Antifungal Activity Toward Fusarium culmorum in Soluble Wheat Extracts

ABSTRACT This study investigated antifungal activity in soluble extracts from seed of a range of wheat cultivars differing in susceptibility to Fusarium head blight. Antifungal activity was assessed in terms of beta-D-glucuronidase (GUS) activity of a Fusarium culmorum GUS transformant using a sensitive laboratory assay. Significant antifungal activity was detected in seed extracts from WEK0609, CM 820036, and Arina. Initial characterization of the Arina seed extract indicated that it contained antifungal proteinaceous compounds. The Arina extract yielded two (60 and 80%) ammonium sulfate fractions containing inhibitory compounds. Gel filtration chromatography and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis of antifungal fractions showed that the antifungal activities detected in the Arina 60 and 80% ammonium sulfate fractions were associated with putative proteinaceous compounds with apparent molecular masses of approximately 60 and 28 kDa, respectively.     

Effects of sterol biosynthesis inhibitor fungicides in the phytopathogenic fungus,Nectria haematococca: ergosterol depletion versus precursor or abnormal sterol accumulation as the mechanism of fungitoxicity

The relative importance of the depletion of ergosterol versus the accumulation of precursor or abnormal sterols in the mechanism of fungal growth inhibition by sterol biosynthesis inhibitor fungicides is incompletely understood. In order to investigate this problem further, the degree of inhibition of the growth of Nectria haematococca by fungicides with different enzymatic targets in the sterol biosynthetic pathway was determined and compared with their effects on the sterol profile. The sensitivity of N. haematococca was highest towards fenpropimorph, followed by tebuconazole, terbinafine, fenpropidin and tridemorph. Terbinafine, a squalene epoxidase inhibitor, induced a very large accumulation of squalene without very significant inhibition of ergosterol biosynthesis and growth. The fungus appeared able to tolerate large amounts of squalene. In the case of tebuconazole, a sterol 14α-demethylase inhibitor, it seemed that the accumulation of C4 mono- and dimethyl sterols was responsible for fungitoxicity. Fenpropimorph and fenpropidin seemed to be good inhibitors of both sterol Δ¹⁴-reductase and Δ⁸→Δ⁷-isomerase, whereas tridemorph was a better inhibitor of Δ⁸→Δ⁷-isomerase than of the Δ¹⁴-reductase. Large accumulations of Δ^8,14- or Δ⁸-sterols and correspondingly large decreases in the ergosterol content are both implicated in the fungitoxicity of these compounds in N. haematococca. © 1998 Society of Chemical Industry     

Molecular basis for the antimycotic and antibacterial activity of N-substituted imidazoles and triazoles: The inhibition of isoprenoid biosynthesis

The antimycotic N-substituted imidazoles and triazoles, such as imazalil, ketoconazole and itraconazole, interfere selectively at low concentrations (≥0.01nm) with the 14α-demethylase system (which is dependent on cytochrome P-450) of fungal cells, for example, Candida albicans and Penicillium italicum. This results in a decreased availability of ergosterol and the accumulation of 14α-methyl-sterols such as lanosterol. Cholesterol synthesis in a subcellular fraction of rat liver, in intact fibroblasts, and in vivo in rat liver, was much less sensitive, for example, to ketoconazole. The imidazole derivatives imazalil, miconazole, ketoconazole and parconazole, and the triazole derivatives propiconazole, terconazole and itraconazole affect the cytochrome P-450 species of microsomal fractions from Saccharomyces cerevisiae and rat liver. Cytochrome P-450 of rat-liver microsomes was much less sensitive to these azole derivatives, in parallel with the lower sensitivity of cholesterol synthesis. Using unilamellar vesicles composed of phosphatidylcholine, phosphatidyl-ethanolamine and diphosphatidylcholine, multilamellar vesicles of dipalmitoylphos-phatidylcholine, and intact S. cerevisiae, it was shown that the substitution of ergosterol by lanosterol leads to functional changes in the membranes. It is speculated that the selective interaction of the azole derivatives with the yeast microsomal cytochrome P-450 leads to the accumulation of 14a-methyl-sterols and results in changes in the permeability of the membranes and leakages. The observed inhibition of growth may have its origin in these changes. Miconazole, ketoconazole and deacylated ketoconazole (R-39519) also affect the growth of Staphylococcus aureus, miconazole being 12.5 and 14 times, respectively, more active than R-39519 and ketoconazole. The greater antibacterial activity of miconazole coincides with its greater inhibition of the biosynthesis of C-55 isoprenoid alcohol and vitamin K. The phosphorylated derivative of C-55 isoprenoid alcohol has functional importance in the biosynthesis of bacterial cell wall and membrane polymers, and the menaquinone vitamin K plays a role in the electron transport of Gram-positive bacteria. The reduced synthesis of these vital compounds may contribute to the antibacterial activity of miconazole.     

Bioactivity of extracts and isolated compounds from Vitex polygama (Verbenaceae) and Siphoneugena densiflora (Myrtaceae) against Spodoptera frugiperda (Lepidoptera: Noctuidae)

The effects of crude extracts, fractions and isolated compounds from Vitex polygama Cham. and Siphoneugena densiflora Berg were evaluated on the development of Spodoptera frugiperda JE Smith, a destructive insect pest of corn and several other crops. The extracts and fractions were incorporated into an artificial diet at 1 mg g(-1) and offered to the insect during its larval stage. Length and viability of larval and pupal stages as well as pupal weight were assessed. Isolated compounds were tested through superficial contamination of the diet at 0.1 mg g(-1). Weight and viability of ten-day-old larvae were determined. Methanolic and hydroalcoholic S. densiflora extracts caused 100% larval mortality, while leaf and fruit hydroalcoholic extracts from V. polygama were the most active. Among the isolated compounds, flavonoids presented the best insecticidal results, and tannins the best larval growth inhibition.     

Inhibitors of juvenile hormone biosynthesis in corpora allata of the cockroach Periplaneta americana (L.) in vitro

The spontaneous biosynthesis of methyl (2E,6E)-(10R)-10,11-epoxy-3,7,11-trimethyldodeca-2,6-dienoate (juvenile hormone III; JH III) in excised corpora allata of Periplaneta americana was inhibited by a number of synthetic prop-2-ynyl ethers and 1,3-benzodioxole derivatives. One structurally diverse group of compounds inhibited only the final biosynthetic enzyme methyl farnesoate epoxidase (EC.1.14.14.–) at low to moderate concentrations, but at higher concentrations, also inhibited methyl farnesoate (MF) formation, causing an accumulation of MF in the concentration range 10 to 100 μM. For a second, more limited, group of compounds, there was close congruence between the inhibition of JH III biosynthesis and that of total ester (MF plus JH III) biosynthesis over their effective inhibitory concentration ranges. In contrast to the first group, there was no accumulation of MF and these compounds evidently inhibited JH III biosynthesis, at the level of either farnesoic acid esterification by O-methyl transferase (EC.2.1.1.–), or at an earlier step in the biosynthetic pathway that remains to be elucidated.     

United States Department of Agriculture-Agricultural Research Service research on pre-harvest prevention of mycotoxins and mycotoxigenic fungi in US crops

Mycotoxins (ie toxins produced by molds) are fungal metabolites that can contaminate foods and feeds and cause toxic effects in higher organisms that consume the contaminated commodities. Therefore, mycotoxin contamination of foods and feeds results is a serious food safety issue and affects the competitiveness of US agriculture in both domestic and export markets. This article highlights research accomplished by Agricultural Research Service (ARS) laboratories on control of pre-harvest toxin contamination by using biocontrol, host-plant resistance enhancement and integrated management systems. Emphasis is placed on the most economically relevant mycotoxins, namely aflatoxins produced by Aspergillus flavus, Link, trichothecenes produced by various Fusarium spp and fumonisins produced by F verticillioides. Significant inroads have been made in establishing various control strategies such as development of atoxigenic biocontrol fungi that can outcompete their closely related, toxigenic cousins in field environments, thus reducing levels of mycotoxins in the crops. Potential biochemical and genetic resistance markers have been identified in crops, particularly in corn, which are being utilized as selectable markers in breeding for resistance to aflatoxin contamination. Prototypes of genetically engineered crops have been developed which: (1) contain genes for resistance to the phytotoxic effects of certain trichothecenes, thereby helping reduce fungal virulence, or (2) contain genes encoding fungal growth inhibitors for reducing fungal infection. Gene clusters housing the genes governing formation of trichothecenes, fumonisins and aflatoxins have been elucidated and are being targeted in strategies to interrupt the biosynthesis of these mycotoxins. Ultimately, a combination of strategies using biocompetitive fungi and enhancement of host-plant resistance may be needed to adequately prevent mycotoxin contamination in the field. To achieve this, plants may be developed that resist fungal infection and/or reduce the toxic effects of the mycotoxins themselves, or interrupt mycotoxin biosynthesis. This research effort could potentially save affected agricultural industries hundreds of millions of dollars during years of serious mycotoxin outbreaks.     

Identification of a Maize Kernel Pathogenesis-Related Protein and Evidence for Its Involvement in Resistance to Aspergillus flavus Infection and Aflatoxin Production

ABSTRACT Aflatoxins are carcinogens produced by Aspergillus flavus and A. parasiticus during infection of susceptible crops such as maize. Several aflatoxin-resistant maize genotypes have been identified and kernel proteins have been suggested to play an important role in resistance. In the present study, one protein (#717), which was expressed fivefold higher in three resistant lines compared with three susceptible ones, was identified using proteomics. This protein was sequenced and identified as a pathogenesis-related protein (PR-10) based on its sequence homology. To assess the involvement of this PR-10 protein (ZmPR-10) in host resistance of maize against fungal infection and aflatoxin production, the corresponding cDNA (pr-10) was cloned. It encodes a protein of 160 amino acids with a predicted molecular mass of 16.9 kDa and an iso-electric point of 5.38. The expression of pr-10 during kernel development increased fivefold between 7 and 22 days after pollination, and was induced upon A. flavus infection in the resistant but not in the susceptible genotype. The ZmPR-10 overexpressed in Escherichia coli exhibited a ribonucleolytic and antifungal activities. Leaf extracts of transgenic tobacco plants expressing maize pr-10 also demonstrated RNase activity and inhibited the growth of A. flavus. This evidence suggests that ZmPR-10 plays a role in kernel resistance by inhibiting fungal growth of A. flavus.     

Inhibition of aflatoxin production in Aspergillus flavus by pyrazolines and pyrazoles

A new series of 1,3,5-triarylpyrazolines, obtained from 3, 4-dichloroacetophenone, were readily dehydrogenated by tetrachloro-o-benzoquinone at room temperature to the pyrazoles. The spectra and fluorescence of the compounds are discussed. The pyrazolines and pyrazoles did not significantly affect the mycelial growth of 19 fungal isolates, but at low concentrations, they inhibited aflatoxin production by Aspergillus flavus.     

Compounds from the subterranean part of Johnsongrass and their allelopathic potential

The allelopathic effects of the exotic invasive weed, Johnsongrass, on Lactuca sativa, a native plant in China, were evaluated and the phytotoxins were investigated under laboratory conditions. The crude extracts (chloroform and ethyl acetate fractions) that were obtained from the ethanol extract of the subterranean parts of Johnsongrass inhibited the germination speed and growth of the roots and shoots of the test plant. The four compounds, ethyl p‐hydroxybenzoate, diosmetin, apigenin, and luteolin, were isolated from the extract of the subterranean parts of Johnsongrass first, along with three other compounds (reported previously), p‐hydroxybenzaldehyde, p‐hydroxybenzoic acid, and dhurrin, and they all were evaluated on L. sativa. At the concentration of <0.5 mmol L^?1, ethyl p‐hydroxybenzoate, dhurrin, and the mixture of the compounds delayed the germination speed of the seeds of L. sativa at 24 h of incubation, while apigenin delayed the germination speed of the seeds of L. sativa at 72 h of incubation. Ethyl p‐hydroxybenzoate demonstrated the strongest delaying effect among the phenols and a similar effect was found with apigenin among the flavonoids, wheras ethyl p‐hydroxybenzoate induced a drastic inhibition of the germination at 2 mmol L^?1. In contrast, p‐hydroxybenzaldehyde and luteolin had no effect on the germination at any concentration at any stage. All the substances inhibited the shoot and root growth of L. sativa at 3 mmol L^?1. Increasing the concentration increased the inhibition of the growth of L. sativa. The inhibitory activity of ethyl p‐hydroxybenzoate and p‐hydroxybenzaldehyde was greater than that of the other compounds. This result suggested that the isolated phytotoxins might contribute to the successful invasion by Johnsongrass.     

Effects of clarified neem oil on growth and aflatoxin B1 formation in submerged and plate cultures of aflatoxigenicAspergillus spp.

An increase of 11–31% of dry mycelial mass was observed along with a slight decrease (5–10%) in aflatoxin Bi production in 5-day-old aflatoxigenicAspergillus spp. submerged cultures containing either 0.5 ml or 1.0 ml clarified neem oil (CNO) in 0.1 % Triton solution. Fungal growth and aflatoxin B₁ production were also determined in potato-dextrose-agar petri plate cultures inoculated with aflatoxigenicAspergillus spp. containing an atmosphere of volatiles emitted from 0.25 ml, 0.5 ml, and 1.0 ml CNO added to the plates. After 5 days’ incubation, fungal radial growth was reduced by 7–29% and aflatoxin B₁ production by 0–67%. GC/MS analysis of the head space volatiles of the CNO indicated that the reduction of fungal growth and aflatoxin B₁ was probably due to low molecular weight hydrocarbons, aldehydes, alcohols, and sulfur compounds emitted at 30°C in the dry culture. These results suggest that volatiles emitted from CNO at 30° C in plate cultures were more fungistatic and consequently inhibited aflatoxin production more than neem oil added in liquid cultures.     

Effects of sterol biosynthesis-inhibiting (SBI) fungicides on cytochrome P-450 oxygenations in fungi

The fungicides miconazole, fenarimol, and etaconazole block ergosterol biosynthesis in fungi by inhibiting sterol 14α-demethylation, which is mediated by a cytochrome P-450 enzyme. The sensitivity of cytochrome P-450-dependent hydroxylation or demethylation of several substrates to these fungicides and similar compounds was compared to that of fungal growth and sterol 14α-demethylation. Demethylation of p-chloro-N-methylaniline (PCMA) by sporidia of Ustilago maydis and 11α-hydroxylation of progesterone by Aspergillus nidulans were relatively insensitive to these compounds and to metyrapone. The ability of a sterol 14α-demethylation-deficient mutant to demethylate PCMA indicates that this substrate is not demethylated by the sterol 14α-demethylation system of U. maydis. The 14α-hydroxylation of progesterone by cells of Curvularia lunata was quite sensitive to the three fungicides, and also to metyrapone and isopropylphenylimidazole. This system was less sensitive to the three fungicides than sterol 14α-demethylation, but was appreciably more sensitive than PCMA demethylation. A study of progesterone 14α-hydroxylation in cell-free preparations of C. lunata showed the reaction to be inhibited by CO, and to be competitively inhibited by low concentrations of miconazole. These data suggest that the primary action of sterol biosynthesis-inhibiting (SBI) fungicides is competitive inhibition of sterol/steroid-type cytochrome P-450 enzymes rather than interference with the function of sterol carrier proteins or enzyme-modulating phospholipids.