No abstract

DNA fingerprinting was performed on 72 strains of Aspergillus oryzae and 9 strains of Aspergillus sojae isolated from chu (China) or koji (Japan) mold inoculum used in the production of traditional Oriental fermented beverages or foods including soy sauce, miso, and sake. The cultures were deposited with the ARS Culture Collection (NRRL) between 1909 and 2001. PstI digests of total genomic DNA from each isolate were probed using the pAF28 repetitive sequence. All strains of A. sojae that we examined produced an identical DNA fingerprint and belong to the same DNA fingerprint group (GTAo-9). Strains of A. oryzae were distributed among 41 DNA fingerprint groups, including GTAo-12 represented by 11 strains, GTAo-19 represented by 5 strains, GTAo-5 and GTAo-15 each represented by 4 strains, and GTAo-8, GTAo-17, and GTAo-24 each represented by 3 strains. Thirty-three single strain isolates of A. oryzae produced unique fingerprints. Our data offer evidence suggesting that (1) the successful domestication of A. parasiticus genotypes yielding A. sojae occurred far less frequently than among genotypes of A. flavus var. oryzae; and (2) some Aspergillus genotypes employed in different fermentations and regions were derived from a common ancestral clonal population.

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. Aspergillus flavus Link is a fungal plant pathogen that infects seeds of corn, cotton, peanuts, and tree nuts and may produce aflatoxins, a serious food safety hazard (10). Aflatoxins are recognized as potent hepatotoxins and carcinogens causing mortality or reducing the productivity of farm animals. Aflatoxin-contaminated foodstuffs have also been associated with a high incidence of liver cancer in humans (4, 32). Our laboratory is studying the environmental conditions that contribute to aflatoxin outbreaks in preharvest corn. One of our objectives is to identify the source(s) of the A. flavus population that infects the grain (e.g., soil, corn insects, crop residues, wind-borne spores, etc.) and to determine if some strains are selectively favored by an ability to infest preharvest corn (6, 8, 31). In order to accomplish this, it is necessary that we identify A. flavus isolates belonging to the same genotype or clone. The ability to characterize and monitor genetically identical strains from A. flavus populations should allow one to determine how the disease is spread and which of the subpopulations are associated with aflatoxin-contaminated seeds at harvest. Repetitive DNA sequences have proven useful and reliable characters in evaluating the genetic relatedness of strains at different levels of classification (e.g., species, forma speciales, vegetative compatibility groups [VCGs], clonal isolates) (7, 12, 18, 21-24, 30). In this report, we describe the construction and characterization of a molecular hyb...