Donald T. Wicklow

Penicillium thiersii, Penicillium angulare and Penicillium decaturense, new species isolated from wood-decay fungi in North America and their phylogenetic placement from multilocus DNA sequence analysis

We describe three new fungicolous species on the basis of phenotypic and phylogenetic differences from known species. Penicillium thiersii, P. angulare and Penicillium decaturense are described. Penicillium thiersii phenotypically is identified on the basis of several characteristics including growth rates, vesicle size and condium shape and roughening. Penicillium angulare is related most closely to P. adametzioides but differs from it by restricted growth rates and conidiophores greater than 60 μm in length. Penicillium decaturense is related most closely to P. miczynskii but differs from that species by growth rate, minimum growth temperature and pigment production on MEA. Multilocus phylogenetic analysis confirmed the genetic distinctiveness of P. decaturense and the closely related species P. miczynskii, P. chrzaszczii and P. manginii. Penicillium rivolii is a synonym of P. waksmanii on the basis of this analysis. Analysis of the EF-1α gene shows rapid changes of position, number and length of introns between the species, suggesting a recent evolutionary origin for the introns.

Aspergillus pseudotamarii, a new aflatoxin producing species in Aspergillus section Flavi

A recent report of an aflatoxin producing isolate of Aspergillus tamarii prompted a taxonomic re-examination of aflatoxigenic and non-aflatoxigenic isolates identified as A. tamarii as well as the closely related A. caelatus. Representatives of each species, including atypical isolates, were compared morphologically, for mycotoxin production, and for divergence in ITS, 28S, β-tubulin and calmodulin gene sequences. Because of genetic, morphological, and mycotoxin differences, the aflatoxin producing isolates of A. tamarii are given species rank as Aspergillus pseudotamarii sp. nov.

DNA relatedness among wild and domesticated species in the Aspergillus flavus group

The aflatoxin-producing fungi Aspergillus flavus and A. parasiticus show many phenotypic similarities with the nonaflatoxigenic species A. oryzae and A. sojae which are widely used in food fermenta...

A protective endophyte of maize: Acremonium zeae antibiotics inhibitory to Aspergillus flavus and Fusarium verticillioides

The maize endophyte Acremonium zeae is antagonistic to kernel rotting and mycotoxin producing fungi Aspergillus flavus and Fusarium verticillioides in cultural tests for antagonism, and interferes with A. flavus infection and aflatoxin contamination of preharvest maize kernels. Chemical studies of an organic extract from maize kernel fermentations of Acremonium zeae (NRRL 13540), which displayed significant antifungal activity against Aspergillus flavus and F. verticillioides, revealed that the metabolites accounting for this activity were two newly reported antibiotics pyrrocidines A and B. Pyrrocidines were detected in fermentation extracts for 12 NRRL cultures of Acremonium zeae isolated from maize kernels harvested in Illinois (4/4 cultures), North Carolina (5/5), Georgia (1/2) and unrecorded locations within the USA (2/2). Pyrrocidine B was detected by LCMSMS in whole symptomatic maize kernels removed at harvest from ears of a commercial hybrid that were wound-inoculated in the milk stage with A. zeae (NRRL 13540) or (NRRL 13541). The pyrrocidines were first reported from the fermentation broth of an unidentified filamentous fungus LL-Cyan426, isolated from a mixed Douglas Fir hardwood forest on Crane Island Preserve, Washington, in 1993. Pyrrocidine A exhibited potent activity against most Gram-positive bacteria, including drug-resistant strains, and was also active against the yeast Candida albicans. In an evaluation of cultural antagonism between 13 isolates of A. zeae in pairings with A. flavus (NRRL 6541) and F. verticillioides (NRRL 25457), A. zeae (NRRL 6415) and (NRRL 34556) produced the strongest reaction, inhibiting both organisms at a distance while continuing to grow through the resulting clear zone at an unchanged rate. Maximum colony diameters for A. zeae (NRRL 6415) and (NRRL 13540), on potato dextrose agar after 14 d, were attained within the range of 25-30 degrees C, with less growth recorded at 15 degrees and 37.5 degrees and no growth at 5 degrees. Potential interactions between A. zeae and other maize endophytes are considered and the significance of these interactions relative to the aflatoxin and fumonisin contamination of preharvest maize is presented. This is the first report of natural products from Acremonium zeae.

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.

DETECTING AFLATOXIN IN SINGLE CORN KERNELS BY TRANSMITTANCE AND REFLECTANCE SPECTROSCOPY

Transmittance spectra (500 to 950 nm) and reflectance spectra (550 to 1700 nm) were analyzed to determine if they could be used to distinguish aflatoxin contamination in single whole corn kernels. Spectra were obtained on whole corn kernels exhibiting various levels of bright greenish–yellow fluorescence. Afterwards, each kernel was analyzed for aflatoxin following the USDA–FGIS Aflatest affinity chromatography procedures. Spectra were analyzed using discriminant analysis and partial least squares regression. More than 95% of the kernels were correctly classified as containing either high (>100 ppb) or low (<10 ppb) levels of aflatoxin. Classification accuracy for kernels between 10 and 100 ppb was only about 25%, but these kernels do not usually affect total sample concentrations and are not as important. Results were similar when using either transmittance or reflectance, and when using either discriminant analysis or partial least squares regression. The two–feature discriminant analysis of transmittance data gave the best results. However, for automated high–speed detection and sorting, instrumentation that uses single–feature reflectance spectra may be more practically implemented. This technology should provide the corn industry with a valuable tool for rapidly detecting aflatoxin in corn.

Reflectance and Transmittance Spectroscopy Applied to Detecting Fumonisin in Single Corn Kernels Infected with Fusarium verticillioides

ABSTRACT Reflectance and transmittance visible and near-infrared spectroscopy were used to detect fumonisin in single corn kernels infected with Fusarium verticillioides. Kernels with >100 ppm and <10 ppm could be classed accurately as fumonisin positive or negative, respectively. Classification results were generally better for oriented kernels than for kernels that were randomly placed in the spectrometer viewing area. Generally, models based on reflectance spectra have higher correct classification than models based on transmittance spectra. Statistical analyses indicated that including near-infrared wavelengths in calibrations improved classifications, and some calibrations were improved by including visible wavelengths. Thus, the color and chemical constituents of the infected kernel contribute to classification models. These results show that this technology can be used to rapidly and nondestructively screen single corn kernels for the presence of fumonisin, and may be adaptable to on-line detection...

Reduction of Aflatoxin and Fumonisin Contamination in Yellow Corn by High-Speed Dual-Wavelength Sorting

ABSTRACT A high-speed dual-wavelength sorter was tested for removing corn contaminated in the field with aflatoxin and fumonisin. To achieve accurate sorting, single kernel reflectance spectra (500–1,700 nm) were analyzed to select the optimal pair of optical filters to detect mycotoxin-contaminated corn during high-speed sorting. A routine, based on discriminant analysis, was developed to select the two absorbance bands in the spectra that would give the greatest classification accuracy. In a laboratory setting, and with the kernels stationary, absorbances at 750 and 1,200 nm could correctly identify >99% of the kernels as aflatoxin-contaminated (>100 ppb) or uncontaminated. A high-speed sorter was tested using the selected filter pair for corn samples inoculated with Aspergillus flavus; naturally infested corn grown in central Illinois; and naturally infested, commercially grown and harvested corn from eastern Kansas (2002 harvest). For the Kansas corn, the sorter was able to reduce aflatoxin levels by ...

ANTIINSECTAN ALKALOIDS : SHEARININES A-C AND A NEW PAXILLINE DERIVATIVE FROM THE ASCOSTROMATA OF EUPENICILLIUM SHEARII

Abstract Four new antiinsectan indole alkaloids ( 1–4 ) have been isolated from organic extracts of the sclerotioid ascostromata of Eupenicillium shearii (NRRL 3324). These extracts also afforded five known, related metabolites ( 5–9 ). The structures of the new compounds were determined through analysis of 1 H NMR, 13 C NMR, HMQC, and HMBC experiments. Compounds 1–9 were isolated from fractions displaying activity in dietary assays against the corn earworm Helicoverpa zea and the dried-fruit beetle Carpophilus hemipterus , and most of the compounds show potent activity in these assays. Shearinine A ( 1 ) also exhibited activity in a topical assay against H. zea , and shearinine B ( 2 ) caused significant mortality in a leaf disk assay against the fall armyworm Spodoptera frugiperda .

Antimicrobial Activity of Pyrrocidines from Acremonium zeae Against Endophytes and Pathogens of Maize

Acremonium zeae produces pyrrocidines A and B, which are polyketide-amino acid-derived antibiotics, and is recognized as a seedborne protective endophyte of maize which augments host defenses against microbial pathogens causing seedling blights and stalk rots. Pyrrocidine A displayed significant in vitro activity against Aspergillus flavus and Fusarium verticillioides in assays performed using conidia as inoculum, with pyrrocidine A being more active than B. In equivalent assays performed with conidia or hyphal cells as inoculum, pyrrocidine A revealed potent activity against major stalk and ear rot pathogens of maize, including F. graminearum, Nigrospora oryzae, Stenocarpella (Diplodia) maydis, and Rhizoctonia zeae. Pyrrocidine A displayed significant activity against seed-rotting saprophytes A. flavus and Eupenicillium ochrosalmoneum, as well as seed-infecting colonists of the phylloplane Alternaria alternata, Cladosporium cladosporioides, and Curvularia lunata, which produces a damaging leaf spot disease. Protective endophytes, including mycoparasites which grow asymptomatically within healthy maize tissues, show little sensitivity to pyrrocidines. Pyrrocidine A also exhibited potent activity against Clavibacter michiganense subsp. nebraskense, causal agent of Gosss bacterial wilt of maize, and Bacillus mojaviense and Pseudomonas fluorescens, maize endophytes applied as biocontrol agents, but were ineffective against the wilt-producing bacterium Pantoea stewartii.