Strain degeneration in Aspergillus parasiticus: A model system for variation in secondary metabolite producing filamentous fungi
Description
Strain variation ('degeneration') in fungi is characterized by changes in phenotype, attenuated virulence, reduced sporulation, and/or loss of secondary metabolite production. In this study, wild type and mutant strains of Aspergillus parasiticus (designated sec+) making aflatoxin and/or pigmented pathway intermediates were subjected to a protocol of serial transfer of mycelial macerates in a defined medium. Variant forms (designated sec$-$) exhibiting altered morphology, scanty sporulation, and lack of detectable amounts of secondary metabolite production were isolated after 5-12 transfers of mycelia. In contrast, when spores rather than mycelia were serially transferred, no $sec-$ forms were generated. The $sec-$ forms were stable and did not revert to sec+ phenotype upon subculturing on complete medium for 10 weeks. The DNA methylation patterns and, with one exception, the dry weights of the sec+ and $sec-$ strains were similar To conduct parasexual cycle analysis, heterokaryons between sec+ and $sec-$ forms with contrasting auxotrophic and spore color markers were produced. Spore color, auxotrophic markers, and the sec+ or $sec-$ phenotype failed to segregate in the heterokaryon test, suggesting that degeneration was not under cytoplasmic control. Qualitative and quantitative assays of heterozygous diploids showed similar aflatoxin production (155-157 $\mu$g/100 ml defined medium) in sec+/sec+ and sec+/sec$-$ diploids, indicating dominance of the sec+ phenotype Haploids were isolated from diploids using benomyl as the haploidization agent. The appearance of a few revertants of the $sec-$ forms from the sec+/sec$-$ crosses indicated that the aflatoxin pathway genes were present but turned off in these variants. To investigate the nature of non-expression, biotransformation experiments were conducted, where both sec+ and sec$-$ forms were fed with 200 $\mu$g of sterigmatocystin, an aflatoxin precursor. All the sec+ forms successfully biotransformed sterigmatocystin to aflatoxin. All the sec$-$ forms did not convert the precursor to detectable amounts of aflatoxin. This phenomenon of non-expression of aflatoxin pathway genes in the experimentally induced sec$-$ forms suggests that this fungus could be developed as a model system to understand the poorly understood process of strain degeneration in filamentous fungi