Impacts of repeated liquid culture on entomopathogenic fungi

ABSTRACT Production of entomopathogenic fungi (EPF) for biopesticides relies on in vitro production of fungal propagules as the active ingredients. Repeated culturing or sub culturing may select for fungi with reduced spore production and virulence. We completed 60 cycles of repeated liquid culture on six isolates representing four genera of EPF. Measurements of the spent media recorded at T2, T15, T30, T45 and T60 cycles included blastospore concentrations, dry matter accumulation as hyphae, glucose concentration remaining, and final pH. Insecticidal activity (LC50 of Trichoplusia ni neonates) was compared between initial and final cultures for conidia grown on nutrient agar. Virulence of Beauveria bassiana GHA was 3× lower at T60 (LC50 ratio = 0.308) and consumption of glucose increased with additional culture cycles. Two Cordyceps javanica (MBC 177 and Apopka 97) had fewer blastospores, higher pH, and altered mycelia dry weight at the T15 sample, but maintained similar values from T15 through T60. MBC 177 and Apopka 97 lost virulence with LC50 ratios of 0.345 and 0.016, respectively. Metarhizium robertsii and M. brunneum F52 isolates failed to produce conidia by plating T30, T45, and T60 cultures on nutrient agar. When comparing T0 with T15 cultures, M. robertsii conidia had increased virulence while M. brunneum had decreased virulence (LC50 ratios of 1.746 and 0.740, respectively). These differences between the two Metarhizium species demonstrate that the direction and level of impact imposed by repeated culture is at least species dependent.