(1099-D) Development and validation of a HTS Platform for the discovery of new antifungal agents against fungal plant pathogens
Tuesday, February 6, 2024
2:00 PM – 3:00 PM EST
Location: Exhibit Halls AB
Abstract: Fungal phytopathogens are the major agents responsible for causing severe damage and losses in agriculture worldwide. Botrytis cinerea, Colletotrichum acutatum, Fusarium proliferatum, and Magnaporthe grisea are included in the top ten fungal phytopathogens that cause important plant diseases on a broad range of crops. Microbial natural products can be an attractive alternative for the biological control of plant pathogens, allowing an economically and environmentally sustainable agriculture. The main goal of this work was to develop and validate a High-Throughput Screening (HTS) platform to evaluate the antifungal potential of chemicals and natural products against these four important plant pathogens.
Several experiments were performed to establish the optimal assay conditions that provide the best reproducibility and robustness for each phytopathogenic strain. For this purpose, the evaluated parameters were two media formulation (SDB and RPMI-1640), several inoculum concentrations (1 x 106, 5 x 105 and 5x106 conidia/mL), the germination curves of each strain, the tolerance to dimethyl sulfoxide (DMSO) and the dose response curve of the antifungal control (Amphotericin B). The assays were performed in 96-well plates, where absorbance at 620 nm was measured before and after incubation to evaluate growth inhibition, and fluorescence intensity at 570 nm excitation and 615 nm emission was monitored after resazurin addition for cell viability evaluation. Quality control parameters (RZ’ Factors, Signal to Background (S/B) ratios and IC50 of the positive control) were determined for each assay batch by Genedata Screener® software. Additional statistical analysis using JMP® software was applied to distribute and categorize the activity population for hit selection.
Once the optimal assay conditions were established for each plant pathogen, a validation of the assay parameters was performed in a primary screening with 40 known fungicide agents (synthetic and natural) belonging to 25 chemical classes with different mode of action. The active compounds were evaluated in dose response curves to determine their IC50 as indicative of the antifungal potential. Finally, the assays were also validated with a small subset of 2400 microbial natural product extracts from the MEDINA Library. Statistical analysis showed that hit selection criterion should consider actives in both techniques (absorbance and fluorescence), providing hit rates of 0.71% for C. acutatum, 1.04% for F. proliferatum, 1.50% for B. cinerea, and 2.63% for M. grisea. Future studies will focus on the characterization of the chemical diversity of new bioactive metabolites detected in this preliminary screening and extending the use of the HTS phytopathogen platform for the discovery of new biocontrol products in plant protection.