Maren A. Klich

Biogeography of Aspergillus species in soil and litter

Based on counts of Aspergillus species reported in over 250 studies of microfungi from soils and litter, chi-square analyses were conducted on species occurrence in five biomes and five latitude ranges to determine variations from expected distributions. There was no overall trend in distribution of the members of the entire genus by biome, however, individual sections of the genus appeared to have distinct distribution patterns. Most members of sections Aspergillus, Nidulantes, Flavipedes and Circumdati occurred at greater than expected frequencies in desert soils. There was no distinct pattern of species occurrence for forest, wetland, or cultivated soils, although members of section Nidulantes were quite rare in cultivated soils. Most species occurred at or below expected frequencies in grassland soils. Members of the genus tended to occur at greater than expected frequencies at latitudes in the subtropical/warm temperate zone between 26 and 35 degrees. Most species occurred at expected frequencies in the lower latitudes, and below expected frequencies in latitudes greater than 35 degrees.

Molecular and physiological aspects of aflatoxin and sterigmatocystin biosynthesis by Aspergillus tamarii and A. ochraceoroseus

Abstract Until recently, only three species (Aspergillus flavus, A. parasiticus and A. nomius) have been widely recognized as producers of aflatoxin. In this study we examine aflatoxin production by two other species, A. tamarii and A. ochraceoroseus, the latter of which also produces sterigmatocystin. Toxin-producing strains of A. tamarii and A. ochraceoroseus were examined morphologically, and toxin production was assayed on different media at different pH levels using thin layer chromatography and a densitometer. Genomic DNA of these two species was probed with known aflatoxin and sterigmatocystin biosynthesis genes from A. flavus, A. parasiticus and A. nidulans. Under the high stringency conditions, A. tamarii DNA hybridized to all four of the A. flavus and A. parasiticus gene probes, indicating strong similarities in the biosynthetic pathway genes of these three species. The A. ochraceoroseus DNA hybridized weakly to the A. flavus and A. parasiticus verB gene probe, and to two of the three A. nidulans probes. These data indicate that, at the DNA level, the aflatoxin and sterigmatocystin biosynthetic pathway genes for A. ochraceoroseus are somewhat different from known pathway genes.

Iturin A: A potential new fungicide for stored grains

The removal of many synthetic fungicides from the market has created a demand for new, environmentally safe fungicides. Iturin A, a cyclic lipopeptide produced byBacillus subtilis, has strong antifungal properties and low mammalian toxicity. To determine the efficacy of this compound as a potential fungicide on stored feed grains, lots of corn, peanuts and cottonseed were treated with varying concentrations of iturin A. The mycoflora of treated seed was assayed along with that of untreated seed and seed treated with fungicides used commercially for planting seed. Fungal species varied considerably in their sensitivity to iturin A. Significant reductions in total mycoflora occurred in most seed lots tested at iturin A concentrations of 50 to 100 ppm.

Characterization of the Aspergillus ochraceoroseus aflatoxin/sterigmatocystin biosynthetic gene cluster

Production of carcinogenic aflatoxins has been reported from members of Aspergillus section Flavi, Aspergillus section Nidulantes and a newly proposed Aspergillus section Ochraceorosei that consists of Aspergillus ochraceoroseus and A. rambellii. Unlike members of section Flavi, A. ochraceoroseus and A. rambellii have been shown to accumulate both aflatoxin (AF) and the aflatoxin precursor sterigmatocystin (ST). Alhough morphologically distinct from A. nidulans, molecular characterization of A. ochraceoroseus AF/ST genes and physiological characteristics of AF/ST production indicated that A. ochraceoroseus is more closely related to A. nidulans than to A. flavus. Knowing that the A. nidulans ST gene cluster is organized differently from the A. flavus AF gene cluster, we determined the genetic organization of the AF/ST biosynthetic cluster in A. ochraceoroseus. Sequencing of overlapping lambda clones and genomic PCR fragments obtained by gene-walking techniques demonstrated that the A. ochraceoroseus AF/ST gene cluster is organized much like the A. nidulans ST gene cluster except that the region from aflN to aflW is located directly upstream of aflC and in reverse orientation such that aflW represents the distal end and aflY the proximal end of the cluster. The A. ochraceoroseus cluster genes demonstrated 62–76% nucleotide identity to their A. nidulans ST cluster gene homologs. Transformation of an A. nidulans aflR mutant with the A. ochraceoroseus aflR restored ST production in A. nidulans transformants. PCR amplification of A. rambellii genomic DNA demonstrated that the AF/ST gene cluster is organized in the same manner as that of A. ochraceoroseus.

Phylogenetic and morphological analysis of Aspergillus ochraceoroseus

Aspergillus ochraceoroseus produces the yellow-gold conidia and other characteristics of Aspergillus subgenus Circumdati section Circumdati. However, this species produces aflatoxin, a secondary metabolite characteristic of some members of subgenus Circumdati section Flavi and sterigmatocystin, a related secondary metabolite usually associated with subgenus Nidulantes sections Nidulantes and Versicolores, as well as members of several other genera. Our morphological data support the placement of A. ochraceoroseus in subgenus Circumdati. Sequence data from A. ochraceoroseus aflatoxin and sterigmatocystin genes aflR and nor-1/stcE, as well as 5.8S ITS and beta tubulin genes, were compared to those of aspergilli in sections Circumdati, Flavi, Nidulantes and Versicolores. In the sequence comparisons, A. ochraceoroseus was related more closely to the species in subgenus Nidulantes than to species from subgenus Circumdati.

Inhibitory effects of gossypol, gossypolone, and apogossypolone on a collection of economically important filamentous fungi.

Racemic gossypol and its related derivatives gossypolone and apogossypolone demonstrated significant growth inhibition against a diverse collection of filamentous fungi that included Aspergillus flavus, Aspergillus parasiticus, Aspergillus alliaceus, Aspergillus fumigatus, Fusarium graminearum, Fusarium moniliforme, Penicillium chrysogenum, Penicillium corylophilum, and Stachybotrys atra. The compounds were tested in a Czapek agar medium at a concentration of 100 μg/mL. Racemic gossypol and apogossypolone inhibited growth by up to 95%, whereas gossypolone effected 100% growth inhibition in all fungal isolates tested except A. flavus. Growth inhibition was variable during the observed time period for all tested fungi capable of growth in these treatment conditions. Gossypolone demonstrated significant aflatoxin biosynthesis inhibition in A. flavus AF13 (B(1), 76% inhibition). Apogossypolone was the most potent aflatoxin inhibitor, showing greater than 90% inhibition against A. flavus and greater than 65% inhibition against A. parasiticus (B(1), 67%; G(1), 68%). Gossypol was an ineffectual inhibitor of aflatoxin biosynthesis in both A. flavus and A. parasiticus. Both gossypol and apogossypolone demonstrated significant inhibition of ochratoxin A production (47%; 91%, respectively) in cultures of A. alliaceus.

Influence of iturin A on mycelial weight and aflatoxin production by Aspergillus flavus and Aspergillus parasiticus in shake culture

Iturin A, a peptidolipid produced byBacillus subtilis, inhibits growth of a large number of fungi. In this study, the effects of iturin A were evaluated on nine isolates ofA. flavus and seven isolates ofA. parasiticus in liquid shake culture. The mycelial dry weight of theA. flavus isolates was not significantly influenced by iturin A, however, there was a significant reduction in mycelial dry weight for two of theA. parasiticus isolates. Aflatoxin production was significantly reduced in five of theA. flavus isolates and three of the six aflatoxigenicA. parasiticus isolates. For the other seven isolates, aflatoxin levels were either unchanged or significantly increased in the presence of iturin A. These results indicate that iturin A does not consistently reduce growth or aflatoxin production of these fungi in pure culture.

Fungal Infections of Fresh-Cut Fruit Can Be Detected by the Gas Chromatography–Mass Spectrometric Identification of Microbial Volatile Organic Compounds

There is a large and rapidly growing market for fresh-cut fruit. Microbial volatile organic compounds indicate the presence of fungal or bacterial contamination in fruit. In order to determine whether microbial volatile organic compounds can be used to detect contamination before fruit becomes unmarketable, pieces of cantaloupe, apple, pineapple, and orange were inoculated with a variety of fungal species, incubated at 25 degrees C, then sealed in glass vials. The volatiles were extracted by headspace solid-phase microextraction and analyzed by gas chromatography-mass spectrometry. Forty-five compounds were identified that might serve as unique identifiers of fungal contamination. Fungal contamination can be detected as early as 24 h after inoculation.

Arabidopsis thaliana as Bioindicator of Fungal VOCs in Indoor Air

Abstract In this paper, we demonstrate the ability of Arabidopsis thaliana to detect different mixtures of volatile organic compounds (VOCs) emitted by the common indoor fungus, Aspergillus versicolor, and demonstrate the potential usage of the plant as a bioindicator to monitor fungal VOCs in indoor air. We evaluated the volatile production of Aspergillus versicolor strains SRRC 108 (NRRL 3449) and SRRC 2559 (ATCC 32662) grown on nutrient rich fungal medium, and grown under conditions to mimic the substrate encountered in the built environment where fungi would typically grow indoors (moist wallboard and ceiling tiles). Using headspace solid phase microextraction/gas chromatography-mass spectrometry, we analyzed VOC profiles of the two strains. The most abundant compound produced by both strains on all three media was 1-octen-3-ol. Strain SRRC 2559 made several terpenes not detected from strain SRRC 108. Using a split-plate bioassay, we grew Arabidopsis thaliana in a shared atmosphere with VOCs from the two strains of Aspergillus versicolor grown on yeast extract sucrose medium. The VOCs emitted by SRRC 2559 had an adverse impact on seed germination and plant growth. Chemical standards of individual VOCs from the Aspergillus versicolor mixture (2-methyl-1-butanol, 3-methyl-1-butanol, 1-octen-3-ol, limonene, and β-farnesene), and β-caryophyllene were tested one by one in seed germination and vegetative plant growth assays. The most inhibitory compound to both seed germination and plant growth was 1-octen-3-ol. Our data suggest that Arabidopsis is a useful model for monitoring indoor air quality as it is sensitive to naturally emitted fungal volatile mixtures as well as to chemical standards of individual compounds, and it exhibits relatively quick concentration- and duration-dependent responses.