Impacts of repeated liquid culture on entomopathogenic fungi

Authors: Behle, Robert; Duffield, Kristin; Dunlap, Christopher

Submitted to: Biocontrol Science and Technology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/22/2023
Publication Date: 5/31/2023
Citation: Behle, R.W., Duffield, K.R., Dunlap, C.A. 2023. Impacts of repeated liquid culture on entomopathogenic fungi. Biocontrol Science and Technology. https://doi.org/10.1080/09583157.2023.2217533.
DOI: https://doi.org/10.1080/09583157.2023.2217533

Interpretive Summary: Fungal pathogens that kill insects are produced by companies to make sustainable and ecologically friendly biopesticides, but currently they represent a small fraction of the pesticide market. One problem with these fungi is their lack of stability when grown commercially in artificial media using continual cultures where they can lose their insecticidal properties. In this research, we grew six fungal strains under 60 cycles of repetitive culture, and observed its impact on fungal metabolism, rates of growth, spore production, and virulence to an insect pest. However, the rates of these changes varied among the strains. Documenting these biological changes was necessary before the next step of research, which will determine specific genetic and metabolic mechanisms associated with fungal stability during production of these beneficial fungi. This knowledge will be used to select among new fungal isolates for those with more stable production and will contribute to improved biopesticide products in the future. The resulting knowledge will support greater adoption of biopesticides over chemical pesticides effectively reducing ecological and health hazards associated with current pest control practices.

Technical Abstract: Production of entomopathogenic fungi (EPF) to serve as biopesticides relies on in vitro production of fungal propagules as the active ingredients. Repeated culturing or sub culturing on solid substrates may select for cultured fungi with unfavorable characteristics, including reduced spore production and virulence against pest insects. Unlike previous reports which have primarily been conducted on solid substrate cultures, we explored the impact of 60 cycles of repeated liquid culture on six isolates representing four genera of EPF. Measurements of the spent media were recorded at T2, T15, T30, T45 and T60 cycles and included spore concentrations, dry matter accumulation representing production of hyphae, glucose concentration remaining, and final pH. Insecticidal activity (LC50) was compared between initial (T0) and final (T60) cultures for conidia grown on nutrient agar plates. LC50 of Beauveria bassiana strain GHA was about 3× greater at T60 (LC50 ratio = 0.308) and consumption of glucose increased with additional culture cycles. The spent media from both isolates of Cordyceps javanica (MBC 177 and MBC 378) had significantly fewer spores, higher pH, and altered mycelia dry weight at the T15 sample, but maintained values of culture measurements from T15 through T60. MBC 177 and MBC 378 lost insecticidal activity (greater LC50 values) with LC50 ratios of 0.345 and 0.016, respectively. Metarhizium robertsii and M. brunneum produced fewer spores in culture (< 6.8 × 107 spore mL-1 culture) compared with the other isolates in this study (>1.5 × 108 spore mL-1). Both Metarhizium isolates failed to produce conidia when T30, T45, and T60 cultures were plated on nutrient agar. When comparing T0 with T15 cultures, M. robertsii conidia had increased virulence to T. ni larvae while M. brunneum had decreased virulence (LC50 ratios of 1.746 and 0.740, respectively). These varied results among the six strains of EPF demonstrate that the direction and level of impact imposed by repeated culture is strain dependent.