Identification and effects of maize silk volatiles on cultures of Aspergillus flavus.

Volatiles generated from corn silks of individual genotypes of maize were found to exhibit differences in biological activities when the volatiles were exposed to 5-day solid cultures of Aspergillus flavus. In inverted potato dextrose-agar Petri plate bioassays, it was found that volatiles emitted from silks of the different maize genotypes had a profound effect on the growth of the fungus and, consequently, aflatoxin production. To determine the underlying cause for this bioactivity, volatiles emitted from the maize silks were trapped on Tenax glass columns and were analyzed by GC-MS. Aflatoxin field-resistant maize genotypes exhibited a larger relative concentration of the antifungal aldehyde, furfural (2-furancarboxaldehyde), when compared to the relative concentrations of the field-susceptible varieties tested. In a closed-container 5-day study, it was observed that fresh 1- and 4-day-old corn silk samples of aflatoxin-resistant maize genotypes emitted higher concentrations of furfural compared to those from susceptible genotypes. This observation probably explains the reason for the bioactivity observed in the in vitro bioassays, and the presence of furfural appears to contribute to a defense mechanism for protecting the developing maize kernel from fungal attack.

Effects of volatile aldehydes from Aspergillus-resistant varieties of corn on Aspergillus parasiticus growth and aflatoxin biosynthesis.

The fungi Aspergillus flavus and Aspergillus parasiticus produce a potent class of hepatocarcinogens known as aflatoxins. Corn-derived volatile compounds have been previously found to affect growth and aflatoxin production in A. flavus. In this study, the effects on A. parasiticus of three corn-derived volatile compounds, n-decyl aldehyde, hexanal and octanal, were measured. These three compounds were previously found to be variably expressed in five Aspergillus-resistant maize strains and three susceptible strains. In this study, A. parasiticus radial growth was restricted least by n-decyl aldehyde and most by octanal. Treatments of 100 microl of both hexanal and octanal were found to completely inhibit radial growth of the fungus using an agar plate assay method. While the volatile compound n-decyl aldehyde had less of an effect on radial growth than the other volatiles, the n-decyl aldehyde treated colonies had a predominance of uniquely aerial hyphae. These colony structures were found to have more complex hyphae and significantly fewer conidiophores than the control and other aldehyde treatments. Furthermore, aflatoxin production by the fungus was reduced by n-decyl aldehyde and hexanal, but was stimulated by octanal. The results presented here indicate that all three volatile compounds reduce radial growth but only n-decyl aldehyde significantly inhibits aflatoxin biosynthesis in A. parasiticus.

The effects of selected cotton-leaf volatiles on growth, development and aflatoxin production of Aspergillus parasiticus.

The fungi Aspergillus flavus and Aspergillus parasiticus produce the hepatocarcinogenic, secondary metabolites, aflatoxins, in cottonseed, corn, peanuts and treenuts. Results have shown that aflatoxigenic strains of A. flavus and A. parasiticus grown in the presence of specific cotton-leaf volatiles exhibit alterations in aflatoxin production accompanied by variations in growth of the fungi. In this study, two alcohols (3-methyl-1-butanol (3-MB) and nonanol) and two terpenes (camphene and limonene) were chosen as representative cotton-leaf volatiles based on the effects they had on fungal growth and/ or aflatoxin production in previous investigations. The morphological effects of volatile exposure were examined in correlation with fungal growth and aflatoxin production. 3-MB-treated samples exhibited a decrease in fungal radial growth which was directly proportional to the volatile dosage. Additionally, 3-MB treatment resulted in loss of mycelial pigmentation and a decrease in sporulation. Limonene and camphene-treated samples yielded negligible differences in radial growth and morphology when compared to unexposed controls. In addition to radial growth inhibition, samples grown in the presence of nonanol demonstrated uniquely aerial hyphae. In comparison to an unexposed control, aflatoxin production increased in cultures exposed to 3-MB but decreased when exposed to the other three volatiles studied.

Identification of the bright-greenish-yellow-fluorescence (BGY-F) compound on cotton lint associated with aflatoxin contamination in cottonseed.

In order to characterize the structure of the bright-greenish-yellow-fluorescence (BGY-F) compound on cotton lint associated with aflatoxin contamination in cotton seed, various in vitro and in vivo natural BGY-F reaction products were prepared. Under similar high pressure liquid chromatography separation with variable wavelength and programmable fluorescence detection (HPLC-UV/FL), combined with atmospheric pressure ionization and mass spectral determinations it was found that the BGY-F reaction products prepared from three preparations: (a) kojic acid (KA) + peroxidase (soybean peroxide or horseradish type VI and type II) + H2O2, or (b) detached fresh cotton locules + KA + H2O2, or (c) attached field cotton locules that were treated with a spore suspension of aflatoxigenic Aspergillus flavus, all resulted in identical chromatographic characteristics, and all exhibited a molecular weight of 282. Further characterization of the BGY-F reaction product with 1H- and 13C-NMR spectroscopic analysis revealed that it was a dehydrogenator dimer of 2 KA, linked through the C-6 positions.

Effects of Neem Leaf Volatiles on Submerged Cultures of Aflatoxigenic Aspergillus parasiticus.

Microbe-free compressed air was passed continuously for a 3-day test period through an enclosed system containing fresh neem leaves; the resultant emitted volatiles were passed over the surface of submerged liquid cultures of a wild-type aflatoxigenic isolate of Aspergillus parasiticus. Aflatoxin determinations for the fungal culture that received neem-derived volatiles, after a 3-day incubation period, resulted in a 90% overall reduction in aflatoxin production and a 51% reduction in fungal biomass when compared with cultures that did not receive neem volatiles. In a separate experiment but in a similarly enclosed system, volatiles from fresh neem leaves were collected on a small Tenax column and were thermally desorbed and cryogenically focused on a capillary gas chromatography column. The neem volatiles were subsequently separated and identified by gas chromatography-mass spectrometry. Sixty-eight compounds were identified by comparison of retention times and mass spectra with either authentic compounds or spectra from a computer-assisted library database of mass spectra. It was found that 10% of the total headspace volatiles were composed of C(3) to C(9) alkenals, which are toxic to aflatoxigenic Aspergillus spp., which could explain the bioactivity that resulted in reduced biomass in the neem-treated cultures.

C(15)H(24) Volatile Compounds Unique to Aflatoxigenic Strains of Aspergillus flavus.

Headspace volatiles from eight strains of Aspergillus flavus (four aflatoxigenic strains and four nonaflatoxigenic strains), grown for 1, 2, 3, 4, 8, and 10 days in submerged cultures, were collected in Tenax GC traps. The traps were desorbed onto a 50-m gas-liquid chromatography capillary column by heat and gas purge from an external direct injector device. The column was interfaced with a mass spectrometer data acquisition system. Peaks were identified by comparing retention times and mass spectra with those obtained from authentic compounds and by using a computer-assisted mass spectral data base. Aflatoxigenic strains of A. flavus produced several C(15)H(24) compounds (e.g., alpha-gurjunene, trans-caryophyllene, and cadinene) which peaked in 3-day cultures and were not present in earlier (1- and 2-day) or later (8- and 10-day) cultures. None of these volatiles were detected in nonaflatoxigenic strains of A. flavus. There was an apparent correlation between the release of C(15)H(24) volatile compounds and the initiation of aflatoxin biosynthesis, and a correlation between decline of aflatoxin synthesis and the disappearance of the C(15)H(24) compounds unique to aflatoxigenic A. flavus also existed.

Effect of C6 to C9 alkenals on aflatoxin production in corn, cottonseed, and peanuts.

The effect on aflatoxin production in Aspergillus flavus-inoculated corn, cottonseed, and peanuts in static culture in the presence of gaseous phase C6 to C9 alkenals was investigated. Aflatoxin B1 production was stimulated in corn at the lowest alkenal concentration (1-microliters level) tested. Aflatoxin B1 was completely eliminated at the highest alkenal concentrations (20-microliters level) tested in both treated corn and cottonseed cultures.

Aflatoxin production in cultures of Aspergillus flavus incubated in atmospheres containing selected cotton leaf-derived volatiles.

Aspergillus flavus, in solid culture, was exposed to individual selected, commercially obtained volatile compounds that were similar to volatile cotton leaf-derived components. The radial growth pattern of A. flavus and the production of aflatoxin were determined on these volatile exposed cultures. The most bioactive compounds C6-C9 alkenals, completely inhibited the growth of the fungus. Unexpected results demonstrated that 3-methyl-1-butanol and 3-methyl-2-butanol inhibited A. flavus growth by 20% but increased production of aflatoxin B1 by 1.5 to 2-fold. Other relationships of growth and aflatoxin production resulting from exposure to the bioactive volatiles are discussed.