Effects of media composition on submerged culture spores of the entomopathogenic fungus, Metarhizium anisopliae var. acridum Part 2: Effects of media osmolality on cell wall characteristics, carbohydrate concentrations, drying stability, and pathogenicity

Abstract

This study evaluates osmolality of a submerged conidia-producing medium in relation to the following spore characteristics: yield, morphology (dimensions and cell wall structure), chemical properties of cell wall surfaces (charge, hydrophobicity, and lectin binding), cytoplasmic polyols and trehalose, and performance (drying stability and pathogenicity). Spore production was increased by the addition of up to 150 g l−1 polyethylene glycol 200 (PEG). Spores from high osmolality medium (HOM spores) containing 100 g l−1 PEG had thin cell walls and dimensions more similar to blastospores than submerged conidia or aerial conidia. However, a faint electron-dense layer separating primary and secondary HOM spores’ cell walls was discernable by transmission electron microscopy as found in aerial and submerged conidia but not found in blastospores. HOM spores also appeared to have an outer rodlet layer, unlike blastospores, although it was thinner than those observed in submerged conidia. HOM spores’ surfaces possessed hydrophobic microsites, which was further evidence of the presence of a rodlet layer. In addition, HOM spores had concentrations of exposed N-acetyl-β-d-glucosaminyl residues intermediate between blastospores and submerged conidia potentially indicating a masking of underlying cell wall by a rodlet layer. All spore types had exposed α-d-mannosyl and/or α-d-glucosyl residues, but lacked oligosaccharides. Similar to blastospores, HOM spores were less anionic than submerged conida. Although HOM spores had thin cell walls, they were more stable to drying than blastospores and submerged conidia. Relative drying stability did not appear to be the result of differences in polyol or trehalose concentrations, since trehalose concentrations were lower in HOM spores than submerged conidia and polyol concentrations were similar between the two spore types. HOM spores had faster germination rates than submerged conidia, similar to blastospores, and they were more pathogenic to Schistocerca americana than submerged conidia and aerial conidia.