DNA fingerprinting analysis of vegetative compatibility groups in Aspergillus caelatus.

Forty-three isolates of Aspergillus caelatus, whose vegetative compatibility groups (VCGs) have been identified, were assessed by DNA fingerprinting using a repetitive sequence DNA probe (pAF28) cloned from A. flavus. Thirteen distinct DNA fingerprint groups or genotypes were identified among the 43 isolates. Twenty-four isolates belonging to VCG 1 produced identical DNA fingerprints and included isolates from the United States and Japan. Four other DNA fingerprint groups had multiple isolates sharing identical fingerprints corresponding to VCGs 2, 3, 12 and 13. Eight of the 13 fingerprint groups corresponding to VCGs 4-11 were represented by a single isolate with a unique fingerprint pattern. These results provide further confirmation that the pAF28 probe can distinguish VCGs of species within Aspergillus section Flavi based on DNA fingerprint patterns and that the probe can be used to estimate the number of VCGs in a sample population. Most of the A. caelatus isolates produced fewer restriction fragments and weakly hybridized with the repetitive DNA probe pAF28 compared to hybridization patterns obtained with A. flavus, suggesting less homology of the probe to A. caelatus genomic DNA.

Identification of Fourier transform infrared photoacoustic spectral features for detection of Aspergillus flavus infection in corn.

Aspergillus flavus and other pathogenic fungi display typical infrared spectra which differ significantly from spectra of substrate materials such as corn. On this basis, specific spectral features have been identified which permit detection of fungal infection on the surface of corn kernels by photoacoustic infrared spectroscopy. In a blind study, ten corn kernels showing bright greenish yellow fluorescence (BGYF) in the germ or endosperm and ten BGYF-negative kernels were correctly classified as infected or not infected by Fourier transform infrared photoacoustic spectroscopy. Earlier studies have shown that BGYF-positive kernels contain the bulk of the aflatoxin contaminating grain at harvest. Ten major spectral features, identified by visual inspection of the photoacoustic spectra of A. flavus mycelium grown in culture versus uninfected corn, were interpreted and assigned by theoretical comparisons of the relative chemical compositions of fungi and corn. The spectral features can be built into either empirical or knowledge-based computer models (expert systems) for automatic infrared detection and segregation of grains or kernels containing aflatoxin from the food and feed supply.

Volatiles from Fusarium verticillioides (Sacc.) nirenb. and their attractiveness to nitidulid beetles.

It is known that sap beetles (Coleoptera: Nitidulidae) can vector the fungus Fusarium verticillioides (Sacc.) Nirenb. (= F. moniliforme Sheldon), which causes an important ear-rot disease in corn and also produces fumonisin mycotoxins. The volatiles produced by this fungus were studied to establish whether they could attract sap beetles. Such an association would suggest more than just an incidental role in transmission of the fungus by the beetles. F. verticillioides consistently produces a blend of five alcohols (ethanol, 1-propanol, 2-methyl-1-propanol, 3-methyl-1-butanol, and 2-methyl-1-butanol), acetaldehyde, and ethyl acetate. Ethanol is the most abundant alcohol. The fungus also produces four phenolic compounds (the most abundant of which is ethylguaiacol), a series of presently unidentified sesquiterpene hydrocarbons, and an unidentified compound that is probably a 10-carbon ketone. Solid-phase microextraction was the key technique used in volatile analysis. The volatile profiles change over time and differ somewhat among fungal strains: The alcohols, aldehyde, and ester always appeared first and were present for each strain. Production of the phenolics lagged by several days, and in some strains these compounds were barely detectable. Volatile production eventually diminished in all strains. All strains were attractive to the sap beetle, Carpophilus humeralis (F.), in wind-tunnel bioassays. Attraction was correlated primarily to the presence of the alcohols, acetaldehyde, and ethyl acetate, rather than to the phenolics. To verify that the identified culture volatiles were responsible for beetle attraction, cultures were quantitatively simulated with synthetic chemicals, and the cultures and corresponding synthetic mixtures were then compared by bioassay. The comparisons were favorable. Volatile emission patterns from cultures were fairly robust with respect to inoculum level or incubation temperature, but some manipulation was possible. For example, after freeze-drying and rehydrating (a rapid simulation of winter/spring conditions), F. verticillioides produced ethyl acetate and other esters at unusually high levels. The fungus produced attractive volatiles following ear inoculation of milk-stage field corn as well as on sterile, mature kernels in the laboratory.

Isoform patterns of chitinase and beta-1,3-glucanase in maturing corn kernels (Zea mays L.) associated with Aspergillus flavus milk stage infection.

Isoform patterns of chitinase and beta-1,3-glucanase of maturing kernels of yellow dent corn (Pioneer 3394) infected with Aspergillus flavus at the milk stage were investigated through polyacrylamide gel electrophoresis (PAGE). Proteins on the sodium dodecyl sulfate (SDS) gel with an apparent molecular mass range of 23-46 kDa were differentially present in the kernels infected with both aflatoxin-producing and non-aflatoxin-producing strains of A. flavus. From in-gel (native PAGE) enzyme activity assays, three bands corresponding to chitinase isoforms and two bands corresponding to beta-1,3-glucanase isoforms were detected in the infected kernels. One chitinase isoform of 29 kDa was present only in the infected kernels, and another one of 28 kDa was present in both infected and noninfected kernels. They were judged to be acidic on the basis of their migration on an acrylamide isoelectric focusing (IEF) gel. For the beta-1,3-glucanase, one isoform of 35 kDa was present in both infected and noninfected kernels, but another one, a 33 kDa isoform, was present only in the infected kernels. Both acidic and basic beta-1,3-glucanase isoforms were detected in the IEF gel. The results of this study are the first to demonstrate patterns of enhanced or inducible proteins in maturing corn kernels in response to A. flavus infection at the milk stage. The results also indicate that only particular isoforms of the two hydrolytic enzymes are involved in the maturing corn kernels infected at the milk stage with A. flavus.

Transient infrared spectroscopy for detection of toxigenic fungi in corn: potential for on-line evaluation.

An urgent need for rapid sensors to detect contamination of food grains by toxigenic fungi such as Aspergillus flavus prompted research and development of Fourier transform infrared photoacoustic spectroscopy (FTIR-PAS) as a highly sensitive probe for fungi growing on the surfaces of individual corn kernels. However, the photoacoustic technique has limited potential for screening bulk corn because currently available photoacoustic detectors can accommodate only a single intact kernel at a time. Transient infrared spectroscopy (TIRS), on the other hand, is a promising new technique that can acquire analytically useful infrared spectra from a moving mass of solid materials. Therefore, the potential of TIRS for on-line, noncontact detection of A. flavus contamination in a moving bed of corn kernels was explored. Early test results based on visual inspection of TIRS spectral differences predict an 85% or 95% success rate in distinguishing healthy corn from grain infected with A. flavus. Four unique infrared spectral features which identified infected corn in FTIR-PAS were also found to be diagnostic in TIRS. Although the technology is still in its infancy, the preliminary results indicate that TIRS is a potentially effective screening method for bulk quantities of corn grain.

Aflatoxin reduction in corn through field application of competitive fungi.

Soil in corn plots was inoculated with nonaflatoxigenic strains of Aspergillus flavus and A. parasiticus during crop years 1994 to 1997 to determine the effect of application of the nontoxigenic strains on preharvest aflatoxin contamination of corn. Corn plots in a separate part of the field were not inoculated and served as controls. Inoculation resulted in significant increases in the total A. flavus/parasiticus soil population in treated plots, and that population was dominated by the applied strain of A. parasiticus (NRRL 21369). In the years when weather conditions favored aflatoxin contamination (1996 and 1997), corn was predominately colonized by A. flavus as opposed to A. parasiticus. In 1996, colonization by wild-type A. flavus was significantly reduced in treated plots compared with control plots, but total A. flavus/parasiticus colonization was not different between the two groups. A change to a more aggressive strain of A. flavus (NRRL 21882) as part of the biocontrol inoculum in 1997 resulted in a significantly (P < 0.001) higher colonization of corn by the applied strain. Weather conditions did not favor aflatoxin contamination in 1994 and 1995. In 1996, the aflatoxin concentration in corn from treated plots averaged 24.0 ppb, a reduction of 87% compared with the aflatoxin in control plots that averaged 188.4 ppb. In 1997, aflatoxin was reduced by 66% in treated corn (29.8 ppb) compared with control corn (87.5 ppb). Together, the data indicated that although the applied strain of A. parasiticus dominated in the soil, the nonaflatoxigenic strains of A. flavus were more responsible for the observed reductions in aflatoxin contamination. Inclusion of a nonaflatoxigenic strain of A. parasiticus in a biological control formulation for aflatoxin contamination may not be as important for airborne crops, such as corn, as for soilborne crops, such as peanuts.

Neural network pattern recognition of photoacoustic FTIR spectra and knowledge-based techniques for detection of mycotoxigenic fungi in food grains.

Fourier transform infrared photoacoustic spectroscopy (FTIR-PAS), a highly sensitive probe of the surfaces of solid substrates, is used to detect toxigenic fungal contamination in corn. Kernels of corn infected with mycotoxigenic fungi, such as Aspergillus flavus, display FTIR-PAS spectra that differ significantly form spectra of uninfected kernels. Photoacoustic infrared spectral features were identified, and an artificial neural network was trained to distinguish contaminated form uncontaminated corn by pattern recognition. Work is in progress to integrate epidemiological information about cereal crop fungal disease into the pattern recognition program to produce a more knowledge-based, and hence more reliable and specific, technique. A model of a hierarchically organized expert system is proposed, using epidemiological factors such as corn variety, plant stress and susceptibility to infection, geographic location, weather, insect vectors, and handling and storage conditions, in addition to the analytical data, to predict Al. flavus and other kinds of toxigenic fungal contamination that might be present in food grains.

DNA Fingerprinting Analysis of Vegetative Compatibility Groups in Aspergillus flavus from a Peanut Field in Georgia.

The ability of species-specific DNA probe pAF28 to correctly match 75 strains of Aspergillus flavus isolated from a peanut field in Georgia with 1 of 44 distinct vegetative compatibility groupings (VCGs) was assessed. Multiple strains belonging to the same VCG typically produced identical DNA fingerprints, with the exception of VCG 17 and VCG 24, which contained strains that showed 83 and 87% similarity, respectively. A. flavus isolates sharing more than 80% of the fragments are recognized as belonging to the same DNA fingerprint group. Each VCG represented by a single isolate produced unique DNA fingerprints. The results provide further evidence that the pAF28 probe is able to distinguish A. flavus VCGs based on DNA fingerprints and can be used to predict the approximate number of VCGs in a sample population. The DNA probe also hybridized strongly and displayed multiple and distinct bands with other species in Aspergillus section Flavi: A. bombycis, A. caelatus, A. nomius, A. pseudotamarii, and A. tamarii. Although individual strains representing Aspergillus spp. in section Flavi produced DNA fingerprints with multiple bands, the banding patterns could not be used to classify these strains according to species.

Genotypic Diversity of Aspergillus parasiticus in an Illinois Corn Field.

Aspergillus parasiticus was isolated from direct platings of soil from a corn field near Kilbourne, Illinois. Soil contained 0.2 to 4.0 CFU of Aspergillus flavus and/or A. parasiticus per g of soil. Sixty isolates of A. parasiticus, each from a separately collected soil sample, were examined for ability to produce sclerotia and aflatoxins, and were subjected to DNA fingerprinting. PstI digests of total genomic DNA from each isolate were probed using the pAF28 repetitive sequence. Among 60 isolates analyzed, 33 (55%) distinct DNA fingerprint groups were identified (each group sharing less than 80% pAF28 band similarity), including 50 distinct genotypes (83%) with less than 100% pAF28 band similarity. A single A. parasiticus fingerprint group represented 13% of the sample population. The 83% genotypic diversity of the A. parasiticus population was equivalent to the 81% genotypic diversity recorded earlier for a population of 31 A. flavus isolates from the same field soil. Sclerotia were produced by 82% of the 50 A. parasiticus genotypes during dark incubation at 25°C. All isolates of A. parasiticus producedaflatoxin B1B2 and G1G2, whereas only 36% of the 31 A. flavus isolates from these soils produced aflatoxins.

Conidium germination rate in wild and domesticated yellow-green aspergilli.

Conidia of domesticated yellow-green aspergilli from strains of Aspergillus oryzae (Ahlburg) Cohn and Aspergillus sojae Sakaguchi and Yamada ex Murakami, used in the preparation of koji inoculum, germinate approximately 3 h sooner than conidia produced by related wild species, Aspergillus flavus Link ex Fr. and Aspergillus parasiticus Speare. There was no consistent relationship between average conidium size and estimated 50% germination time. Germination trials were conducted on Czapek agar at 28 degrees C. A hypothesis is offered that, in the propagation of koji inoculum, selection has favored those individuals capable of rapid conidium germination and germ tube extension, attributes that enable them to gain the available substrate during intraspecific competition.

Degradation of lignin by cyathus species.

The ability of 12 Cyathus species to degrade C-labeled lignin in kenaf was studied. The sum of C released into solution plus C released into the gas phase over a 32-day fermentation period was used to determine average daily rates of lignin biodegradation. Cyathus pallidus. C. africanus, and C. berkeleyanus delignified kenaf most rapidly. C. canna showed the greatest preference for lignin degradation over other plant components, and its rate of lignin degradation was only slightly lower than the three most active species. The apparent ability of fungi to metabolize low-molecular-weight lignin breakdown products correlated well with their overall delignification rates. C. stercoreus metabolized degradation products of lignin from wheat straw better than those from kenaf lignin, based on the amount of low-molecular-weight products left in solution.

Interaction of competing fungi with fly larvae.

Saprophytic fungi have degradative abilities and interspecific interactions which suggest that resource use and yield should increase as species number increases, but previous studies show the opposite. As a test of the possibility that invertebrate activity changes fungal resource use patterns, we grew coprophilous fungi on rabbit feces at the same initial density singly or in mixtures of 2, 4, or 6 species, with or without activity of larvalLycoriella mali (Diptera: Sciaridae). Fungi in mixtures without larvae caused less weight loss in one mixture, and greater weight loss in 2 mixtures than when growing alone; fungi in 4 of 6 mixtures produced fewer spores than when growing alone. Overall, without larvae, weight loss did not increase as number of fungal species increased. Larvae did not change the pattern of weight loss or proportions of spores caused by mixing fungal species. Numbers of larvae surviving to pupate rose as fungal species numbers increased; as a result, weight loss increased with fungal species number in cultures with larvae.

Evidence of a competitive hierarchy among coprophilous fungal populations.

Evidence is presented that interference competition may be important in later states of fungal colonization of cattle feces from a semiarid grassland in Colorado. Cultural antagonism was examined among fungal isolates representing early sporulating colonists (Ascobolus furfuraceus and Saccobolus truncatus), later sporulating colonists (Iodophanus carneus, Coniochaeta discospora, Hypocopra merdaria, and Poronia punctata), and one early successional species that is able to persist (sporulate) through later stages (Podospora decipiens). Poronia punctata, a comparatively slower-growing and later-appearing colonist (18- to 54-month-old fecal pats), is uniformly antagonistic to all of seven earlier-appearing and co-occurring fungal species. Antibiosis is believed to account for the observed antagonism. The authors suggest that the evolutionary product of interference competition among coprophilous fungal populations may be a pattern of competitive hierarchy in which certain slower-growing, later-successional species can limit the reproductive potential of other fungal colonists on fecal substrates.

Multispecies interactions affect cytoplasmic incompatibility in Tribolium flour beetles.

Previous studies established that cytoplasmic incompatibility in the confused flour beetle, Tribolium confusum, is mediated by a maternally inherited rickettsia-like bacteria: crosses between infected males and uninfected females are sterile. All other crosses are fertile, and individuals can be cured of infection by treatment with tetracycline. We report that a third species-either actinomycete bacteria Streptomyces or fungi Penicillium-can cure beetles of infection with these parasites. Progeny from infected beetles were raised on flour produced from Streptomyces- or Penicillium-"molded" wheat grain. Microbial strains included known producers of tetracyclines and strains of related species that are commonly isolated from whole or milled grain. High rates of curing, eight of 10 males cured and nine of 10 females cured, were produced with grain molded with Streptomyces aurepfaciens (Northern Regional Research Laboratories [NRRL] 2209), a common soil-inhabiting, tetracycline-producing actinomycete bacterium. Low rates of curing were recorded for Streptomyces griseus (NRRL B-2249; 1/10 females), Penicillium verrocosum (NRRL 3712; 1/20 females), and Penicillium aurantiogriseum var. polonicum (NRRL 3704; 1/20 females). No curing was recorded for infected populations raised on eight other Streptomyces strains, 11 Penicillium strains, an autoclaved control, or brewer's yeast control. The high rate of cures from one strain of actinomycete and low rate from three other strains of fungi and actinomycetes suggest that local "patches" of antibiotic contaminated grain can promote a polymorphism of infection among Tribolium populations in nature.

Factors influencing the inhibition of aflatoxin production in corn by Aspergillus niger.

Aspergillus niger, a mold commonly associated with Aspergillus flavus in damaged corn, interferes with the production of aflatoxin when grown with A. flavus on autoclaved corn. The pH of corn-meal disks was adjusted using NaOH-HCl, citric acid-sodium citrate, or a water extract of A. niger fermented corn. Aflatoxin formation was completely inhibited below pH 2.8-3.0, irrespective of the system used for pH adjustment. When grown in association with A. flavus NRRL 6432 on autoclaved corn kernels, A. niger NRRL 6411 lowered substrate pH sufficiently to suppress aflatoxin production. The biodegradation of aflatoxin B1 or its conversion to aflatoxin B2a were eliminated as potential mechanisms by which A. niger reduces aflatoxin contamination. A water extract of corn kernels fermented with A. niger caused an additional inhibition of aflatoxin formation apart from the effects of pH.

Intrafungal distribution of aflatoxins among conidia and sclerotia of Aspergillus flavus and Aspergillus parasiticus.

This research examines the distribution of aflatoxins among conidia and sclerotia of toxigenic strains of Aspergillus flavus Link and Aspergillus parasiticus Speare cultured on Czapek agar (21 days, 28 degrees C). Total aflatoxin levels in conidia and sclerotia varied considerably both within (intrafungal) and among strains. Aspergillus flavus NRRL 6554 accumulated the highest levels of aflatoxin (conidia: B1, 84000 ppb; G1, 566000 ppb; sclerotia: B1, 135000 ppb; G1, 968000 ppb). Substantial aflatoxin levels in conidia could place at risk those agricultural workers exposed to dust containing large numbers of A. flavus conidia. Cellular ratios of aflatoxin B1 to aflatoxin G1 were nearly identical in conidia and sclerotia even though levels of total aflatoxins in these propagule types may have differed greatly. Aflatoxin G1 was detected in sclerotia of all A. flavus strains but in the conidia of only one strain. Each of the A. parasiticus strains examined accumulated aflatoxin G1 in both sclerotia and conidia. These results are examined in the context of current evolutionary theory predicting an increase in the chemical defense systems of fungal sclerotia, propagules critical to the survival of these organisms.

Biodiversity of yeasts from Illinois maize.

Microflora in wound sites of preharvest maize (including bacteria, yeasts, and filamentous fungi) may play a role in attracting insects to maize plants and may also interact with growth and mycotoxin production by filamentous fungi. As little data are available about the yeasts occurring on maize from the U.S. corn belt, samples of milled maize from experimental plantings at the University of Illinois River Valley Sand Field were analyzed. Yeast counts showed slight yearly fluctuation and varied between 3.60 and 5.88 (log cfu/g maize). The majority of the yeasts were Candida guilliermondii (approximately 55%), Candida zeylanoides (24%), Candida shehatae (11%), and Debaryomyces hansenii (3%). Also present were Trichosporon cutaneum, Cryptococcus albidus var. aerius, and Pichia membranifaciens. The occurrence of killer yeasts was also evaluated. Killer yeasts were detected in maize for the first time and were identified as Trichosporon cutaneum and Candid zeylanoides. These were able to kill some representative yeasts isolated from maize, including Candida guilliermondii, Candida shehatae, and Cryptococcus albidus var. aerius. Other maize yeasts (Candida zeylanoides, Debaryomyces hansenii, Pichia membranifaciens) were not affected. The majority of yeasts found on maize were unable to ferment its major sugars, i.e., sucrose and maltose. Some (e.g., Candida zeylanoides) were not even able to assimilate these sugars. The importance of these properties in relation to insect attraction to preharvest ears of maize is discussed.

Decomposition of Lignocellulose by Cyathus stercoreus (Schw.) de Toni NRRL 6473, a "White Rot" Fungus from Cattle Dung.

Cyathus stercoreus (Schw.) de Toni NRRL 6473, isolated from aged and fragmented cattle dung collected from a Michigan pasture, effected substantial losses in lignin (45%) from wheat straw during a 62-day fermentation (25 degrees C). The basidiomycete also improved wheat straw digestibility by freeing alpha-cellulose for enzymatic hydrolysis to glucose (230 mg of glucose per 1,000 mg of fermented residue). The rationale for selecting C. stercoreus in attempting to biologically modify the lignin and cellulose components in wheat straw or other gramineous agricultural residues was based on the expectation that this organism is ecologically specialized to enzymatically attack the substructures of native lignins in grasses.