Lyophilization and Field Survival of Epiphytic Yeasts from the Phyllosphere of Native Cacao in the Peruvian Amazon

Epiphytic yeasts are promising biocontrol agents of plant diseases but preserving and transferring them to the field is challenging. Here, we studied six cost-effective lyophilization protective agents to preserve seven strains of Amazonian yeast species isolated from the phyllosphere of native cacao (Theobroma cacao) in Peru. We evaluated the viability of yeasts at 30 and 90 days post-lyophilization in vitro, and their survival after controlled inoculation on cacao fruits in the field. The best protective agents were maltodextrin, honey + skim milk, and honey. Wickerhamomyces anomalus KLG-014 and Wickerhamomyces sp. EGZ-38 showed higher than 97.3% viability after 30 days when lyophilized with maltodextrin. Additionally, Candida sp. KLG-103 showed a viability greater than 50% after 30 days when lyophilized with honey + skim milk. At 90 days, W. anomalus KLG-014, Hannaella theobromatis KLG-063, and Kwoniella heveanensis EGZ-07 showed a viability greater than 20%, with the latter showing an outstanding 100% viability, when lyophilized with honey + skim milk. Conversely, sodium alginate was the least protective agent, as yeast showed 0% viability. In the field, W. anomalus KLG-014, K. heveanensis EGZ-07, Debaryomyces hansenii EGZ-31, and Wickerhamomyces sp. EGZ-38 were successfully re-isolated from the surface of cacao fruits under all treatments after 30 days, except for sodium alginate. This was corroborated via morphological and molecular evidence. This study demonstrates that maltodextrin, honey, and skim milk are suitable for ensuring the in vitro viability of biocontrol yeasts up to 90 days after lyophilization, and their survival up to 30 days after inoculation on cacao fruits in the field. This is a first step toward the development of a biocontrol alternative to mitigate cacao pathogens using native microorganisms from the Amazon in Peru.