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.

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.

Toxigenic strains of Fusarium moniliforme and Fusarium proliferatum isolated from dairy cattle feed produce fumonisins, moniliformin and a new C21H38N2O6 metabolite phytotoxic to Lemna minor L.

Corn samples suspected of causing refusal-to-eat syndrome in dairy cattle were examined mycologically. Fusarium moniliforme (14 isolates) and F. proliferatum (12 isolates) were the predominant fungi present. These isolates were tested for mycotoxin production on rice at 25 degrees C. Each strain of F. moniliforme produced fumonisin B1 (FB1: 378-15,600 ppm) and fumonisin B2 (FB2: 2-1050 ppm). Each strain of F. proliferatum produced moniliformin (45-16,000 ppm), FB1 (27-6140 ppm), and FB2 (5-1550 ppm). In addition, a new Fusarium metabolite of molecular composition C21H38N2O6 was produced by 10 of the F. moniliforme isolates and 7 of the F. proliferatum isolates. The metabolite's 1H- and 13C-NMR, HRFAB/MS and IR spectra indicate an alpha amino acid. It is toxic to Lemna minor L. duckweed (LD50 100 micrograms/mL).

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.

Construction and characterization of a DNA probe for distinguishing strains of Aspergillus flavus.

Repetitive DNA sequences have proven useful and reliable characters in evaluating genetic relatedness of strains at different levels of taxonomic classification. A DNA probe was constructed to distinguish among strains of Aspergillus flavus by DNA fingerprinting techniques. Chromosomal DNA of A. flavus var. flavus NRRL 6541 was partially digested with EcoRI and ligated to a Lambda Dash bacteriophage vector. Four lambda clones were identified which displayed multiple and distinct bands when hybridized with chromosomal DNA from seven strains of A. flavus var. flavus digested with either EcoRI or PstI. One of these clones was chosen for further analysis and was subcloned into pUC19. The subclone, pAF28, contained a 6.2-kb chromosomal DNA insert and was able to distinguish among strains characterized by K. E. Papa (Mycologia 78:98-101, 1986) as belonging to 22 different vegetative compatibility groups. The subclone identified unique banding patterns when hybridized to genomic DNA digested with PstI. The cloned probe may be species specific as it hybridized with the DNA of all isolates of A. flavus tested in addition to strains recognized as varieties of A. flavus (e.g., A. flavus var. oryzae, A. flavus var. parasiticus, and A. flavus var. sojae). pAF28 hybridized to a single band on a Southern blot with Aspergillus nomius DNA but did not hybridize with the DNA of other fungal species tested including Aspergillus ochraceus, Aspergillus auricomus, Aspergillus alliaceus, Fusarium moniliforme, and Penicillium thomii.