In Vitro Evaluation of Effectiveness of Antifungal Agents Against Macrophomina phaseolina

Antifungal agents provide both preventive and curative effects by protecting plant surfaces and eliminating existing infections within plant tissues. Evaluating their impact against phytopathogenic fungi is critical for sustainable agriculture. Macrophomina phaseolina is a highly destructive soil-borne pathogen with a broad host range, causing charcoal rot and related diseases that result in severe yield losses in crops such as soybean, sorghum, maize, and legumes. Several methods are employed to assess the in vitro antifungal efficacy of various substances, including broth microdilution, disk diffusion, gradient screening, and agar-based screening. However, these conventional techniques are generally slower, more labor-intensive, and limited by reproducibility issues. The adoption of microplate reader-based antifungal efficacy testing has enhanced efficiency and scalability, enabling high-throughput evaluation of multiple antifungal agents across a range of concentrations while providing a streamlined platform for preliminary screening. This chapter presents a convenient and cost-effective protocol for assessing the activity of antifungal agents against fungi that produce microsclerotia, with emphasis on standardization and quality control measures. The antifungal efficacy testing and cell death assay are employed to determine inhibitory concentrations via spectrophotometry and to quantify hyphal death through fluorescence microscopy, respectively. In addition, detailed procedures for inoculum preparation, antifungal agent dilution, incubation conditions, and statistical analysis are described.