Morphological Transitions Governed by Density Dependence and Lipoxygenase Activity in Aspergillus ﬂavus (cid:1) †

Aspergillus ﬂavus differentiates to produce asexual dispersing spores (conidia) or overwintering survival structures called sclerotia. Results described here show that these two processes are oppositely regulated by density-dependent mechanisms and that increasing the cell density (from 10 1 to 10 7 cells/plate) results in the lowest numbers of sclerotial and the highest numbers of conidial. Extract from spent medium of low-cell- density cultures induced a high-sclerotium-number phenotype, whereas high-cell-density extract increased conidiation. Density-dependent development is also modiﬁed by changes in lipid availability. Exogenous linoleic acid increased sclerotial production at intermediate cell densities (10 4 and 10 5 cells/plate), whereas oleic and linolenic acids inhibited sclerotium formation. Deletion of Af lox encoding a lipoxygenase (LOX) greatly diminished density-dependent development of both sclerotia and conidia, resulting in an overall increase in the number of sclerotia and a decrease in the number of conidia at high cell densities (>10 5 cells/plate). Af lox mutants showed decreased linoleic acid LOX activity. Taken together, these results suggest that there is a quorum-sensing mechanism in which a factor(s) produced in dense cultures, perhaps a LOX-derived metabolite, activates 29°C dark days. sclerotia, conidia and mycelia. The sclerotia were collected and lyophilized, and sclerotium production was determined gravimetrically (mg [dry weight] per plate). Numbers of conidia were determined only for the plates containing 10 and 7 conidia/plate treated with the low-density (10 3 conidia/plate), high-density (10 7 conidia/plate), and medium extracts at 3 days. Conidia by using assay conidial and sclerotial dependence using four replicates for each The entire experiment repeated twice in order to examine reproducibility. Extracts low- and high-density plates and for each experiment. acid assay. Biomedi-cals, were dissolved 0.1 mg/60 (cid:4) on 12.5-mm-diameter ﬁlter paper disks. disk (cid:4) was used as the the were laid on agar surfaces after plates were inoculated with conidia/plate. were incubated in the dark inside cardboard (resulting in continuous darkness) for 7 days. To visualize the sclerotia, plates 70% conidia and aerial mycelia. Sclerotia were freeze-dried, and sclerotium gravimetrically (mg [dry weight] per plate). All of the fatty acid tests with wild-type strain 3357 were performed by using three replicates, the entire experiment was repeated twice, and similar results were obtained. blotting, hybridization, and probe preparation were performed by using standard methods (46). To examine Af lox expression, cultures of wild-type strain NRRL 3357 and complemented strains TSHB3.1C and TSHB3.5C were cultured in shaken liquid GMM containing 2% yeast extract for 48 h at 29°C and 250 rpm. Equal amounts of mycelium were then removed, placed in liquid GMM containing 2% sorbitol, and grown for an additional 2 h with shaking 250 rpm at 29°C; this was followed by harvesting. Three separate repetitions of this experiment yielded similar results. Af lox expression was also assessed by growing strain NRRL 3357, (cid:5) lox strain TSHB2.39, and strain TSHB3.5C in liquid GMM containing 2% sorbitol for 24 h at 29°C and 220 rpm and then transferring the mycelia to ﬁlter paper on solid GMM containing 2%