Characterization of experimentally induced, nonaflatoxigenic variant strains of Aspergillus parasiticus

Abstract

Six previously isolated, nonaflatoxigenic variants of Aspergillus parasiticus, designated sec mutants, were characterized morphologically by electron microscopy, biochemically by biotransformation studies with an aflatoxin precursor, and genetically by Northern (RNA) hybridization analysis of aflatoxin biosynthetic gene transcripts. Scanning electron micrographs clearly demonstrated that compared with the parental sec ؉ forms, the variant sec forms had an abundance of vegetative mycelia, orders of magnitude reduced number of conidiophores and conidia, and abnormal metulae. Conidiospores were detected in sec cultures only at higher magnifications (؋500), in contrast to the sec ؉ (wild-type) strain, in which abundant conidiospores (masking the vegetative mycelia) were observed even at lower magnifications (؋300). All sec ؉ forms, but none of the sec forms, showed bioconversion of sterigmatocystin to aflatoxins. Northern blots probed with pathway genes demonstrated lack of expression of both the aflatoxin biosynthetic pathway structural (nor-1 and omtA) and regulatory (aflR) genes in the sec forms; PCR and Southern hybridization analysis confirmed the presence of the genes in the sec genomes. Thus, the loss of aflatoxigenic capabilities in the sec form is correlated with alterations in the conidial morphology of the fungus, suggesting that the regulation of aflatoxin synthesis and conidiogenesis may be interlinked.

Effect of Zataria multiflora Boiss. Essential Oil on Growth and Citrinin Production by Penicillium citrinum in Culture Media and Mozzarella Cheese

“…At 200 ppm, the inhibition of citrinin production is relatively greater than the inhibition of growth of the fungus, but has not been observed at lower concentrations. It has been suggested that the regulation of toxin synthesis and conidiogenesis may be interlinked (Kale et al 1996).…”

expressed sequence tags for identification of genes with putative roles in aflatoxin contamination of crops

“…The liquid cultures were incubated at 30°C with constant shaking at 150 RPM. The mycelia were harvested by filtration through miracloth at five different time points (18,24,36,48,72 and 96 h) following inoculation with a conidial suspension. The harvested mycelial samples (wet weight 20 g from wheat bran samples and 10 g from each of the rest of the samples) were mixed and frozen in liquid nitrogen for RNA purification.…”

“…Secondary metabolism is often correlates with fungal developmental processes such as sporulation and sclerotia formation [16,18,28]. Mutants that are deficient in sporulation are unable to produce aflatoxins [18,28].…”

“…Secondary metabolism is often correlates with fungal developmental processes such as sporulation and sclerotia formation [16,18,28]. Mutants that are deficient in sporulation are unable to produce aflatoxins [18,28]. A critical advance in this regard was the finding that the regulation of sporulation and ST production is by means of a shared Gprotein mediated growth pathway in A. nidulans [19,29].…”

“…Genetic studies on aflatoxin biosynthesis in A. flavus and A. parasiticus led to the cloning of 25 clustered genes within a 70 kb DNA region responsible for the enzymatic conversions in the aflatoxin biosynthetic pathway [8,9]. Regulatory elements such as aflR [10,11] and aflS (aflJ) [12,13], nutritional and environmental factors [14,15], fungal developmental and sporulation [16][17][18][19] were also found to affect aflatoxin formation. In A. flavus there are eight chromosomes with an estimated genome size of about 33-36 Mbp that harbor an estimated 12,000 functional genes (reviewed in [7,20], Dr. Machida, personal communication).…”