A Rapid Method for Measuring In Vitro Growth in Entomopathogenic Fungi

SIMPLE SUMMARY: This study aimed to develop a rapid and efficient method for measuring the growth of entomopathogenic fungi, which are used as environmentally friendly alternatives to chemical insecticides. The traditional methods used to measure fungal growth are time-consuming and limited in their ability to capture important aspects of growth. In this research, we implemented an indirect measure using a microplate reader, which uses the optical density of small-volume cultures to estimate fungal growth. We directly related changes in optical density to the amount of fungal biomass, and compared the results of this method with traditional measurements on solid agar plates. We found that the microspectrophotometric approach provided accurate and reliable measurements. Our findings revealed differences in growth rates and biomass production among different species and isolates of the fungi. This technique offers a valuable tool for studying the growth dynamics of entomopathogenic fungi, and has practical applications in assessing their virulence and pathogenic potential. It can contribute to our understanding of how these fungi grow during infection, and aid in the development of effective biological control strategies against insect pests. ABSTRACT: Quantifying the growth of entomopathogenic fungi is crucial for understanding their virulence and pathogenic potential. Traditional methods for determining growth, such as biomass determination or colony growth area, are time-consuming and quantitatively and spatially limited in scope. In this study, we introduce a high-throughput method for rapidly measuring fungal growth using spectrophotometry in small-volume, liquid media cultures in 96-well microplates. Optical density (OD) changes were directly correlated with dry weight of samples for six isolates from three species of the genus Metarhizium to validate spectrophotometric growth measurements, and investigate species- and isolate-specific effects. We quantified fungal biomass from the microcultures by extracting, drying, and weighing mycelial mats. From the relationship established between OD and biomass, we generated standard curves for predicting biomass based on the OD values. The OD measurements clearly distinguished growth patterns among six isolates from three Metarhizium species. The logistic growth phase, as captured by the OD measurements, could be accurately assessed within a span of 80 h. Using isolates of M. acridum, M. brunneum, and M. guizhouense, this technique was demonstrated to be an effective, reproducible, and simple method for rapidly measuring filamentous fungal growth with high precision. This technique offers a valuable tool for studying the growth dynamics of entomopathogenic fungi and investigating the factors that influence their growth.