Effects of Water Activity and Temperature on Entomopathogenic Fungal Culture, Implications on Conidia Integrity and Virulence

Effects of available water for microbial growth, water activity (aw) and incubation temperature on qualities of conidia produced by three entomopathogenic fungi, Beauveria bassiana, Isaria farinosa and Metarhizium anisopliae were evaluated in vitro. Sabouraud Dextrose Agar (SDA) (Sigma-Aldrich, 0.995 aw) and SDA containing calculated amounts of glycerol were prepared to create different osmotically-stressed media (aw = 0.98 or 0.96) and poured into 9 cm Petridishes. Agar plugs of seven days old cultures were transferred into the plates, sealed with parafilm and incubated at three different temperatures, 25, 30 and 35°C for 14 days. Harvested conidia from cultures grown at the three levels of interacting aw and temperatures were described in relation to the growth conditions; A1-, A2- and A3-conidia, interacting growth conditions being 0.995 aw x 25°C, 0.995 aw x 30°C and 0.995 aw x 35°C respectively. Similarly, B1-, B2- and B3-conidia (0.98 aw x 25-35°C) and C1-, C2- and C3-conidia (0.96 aw x 25-35°C). The conidia were tested for germination on different osmotically stressed media and virulence was evaluated using Galleria-model mortality bioassay. Germination rates of conidia was based on a 24-hour incubation period at 25°C while virulence was measured by the median lethal time (LT50) against Galleria mellonella larvae. There were significant variabilities in germination rates and virulence in relation to incubation temperature, osmotic status of media and the isolates. The A3-conidia of Beauveria bassiana and those grown on modified media, B2- and B3-conidia, failed to germinate after 24 hours of incubation, and they were less virulent. Similarly, B3- and C3-conidia of M. anisopliae could not germinate after 24 hours. In contrast, all I. farinosa propagules germinated, although the non osmotically-stressed A1-, A2- and A3-conidia had relatively higher germination rates. Generally, the non-osmotically stressed conidia performed better in terms of germination and virulence. This is a model study on the optimal culture conditions for production of conidia of the entomopathogens in Submerged Fermentation (SF) systems.