Bioassay methods for extraction and detection of antifungal potential of the epicuticular layer of Theobroma cacao pods against the witches’ broom pathogen

The witches’ broom disease of cacao, caused by the fungus Moniliophthora perniciosa, is among the main diseases of cacao (Theobroma cacao L.), one of the most important cultures in the tropics, for which chemical control is not very effective. We suggested that the epicuticular wax layer of cacao pods might have antifungal activity against Moniliophthora perniciosa. The objective of this article was to establish a protocol for extraction of the epicuticular layer of cacao pods and establish a bioassay to evaluate the in vitro antifungal activity of these epicuticular extracts on germination of M. perniciosa. First, we standardized the extraction protocol, and extracts of the epicuticular layer from standard, susceptible, and resistant cacao clones to WBDC were obtained. Then, we used an in vitro agar bioassay to evaluate the antifungal activity of the extracts against M. perniciosa basidiospores. The inhibition of the germination was highest (87.2%) for spores treated with the extracts from the resistant genotype. Treatment with extracts from ICS1 (susceptible genotype) did not significantly influence spore germination. We successfully extracted T. cacao wax and suggested that the epicuticular layer of cacao genotypes has antifungal molecules with inhibitory activity against the germination of M. perniciosa basidiospores in vitro.