Antagonistic potential of Wickerhamomyces anomalus against phytopathogens causing Olive Anthracnose and Chestnut Ink Disease

Abstract

Olive Anthracnose (OA) and Chestnut Ink Disease (CID), caused by eukaryotic organisms such as fungi and oomycetes, pose significant challenges to agricultural production, causing severe yield losses and reduced quality of harvested products. Currently, chemical pesticides are widely used to control these pathogens. However, this approach can be expensive and ineffective, and may have detrimental environmental and health effects. Thus, the development of new, greener and more effective strategies is vital for maintaining agricultural productivity and mitigating the impacts of plant diseases and plagues on food supply and economy on Portugal and other producing countries. The use of yeasts as biological control agents is an effective and environmentally friendly alternative approach for controlling plant pathogens. In this work, we explore the potential of Wickerhamomyces anomalus as a living biocontrol agent for the sustainable pre-harvest control of OA and CID, and to unveil its mode of action. We conducted antagonism assays in liquid media, co-culturing the yeast W. anomalus with three strains of Colletotrichum, causing OA, and two strains of Phytophthora, causing CID. The mode of action of W. anomalus was assessed in several aspects. (i) The viability of the mycelia resulting from the co-cultures was evaluated through re-culturing the mycelia and by microscopic inspection of Methylene Blue-staining mycelia. (ii) Additionally, two common biocontrol strategies were tested - the secretion of cell-wall degrading enzymes and siderophores. (iii) Finally, the interactions between yeast and phytopathogens were further investigated through SEM microscopy. Results confirmed the strong ability of W. anomalus to inhibit the growth of both Colletotrichum and Phytophthora strains, although in different manners. We found that (i) the fungal cells were stained with Methylene Blue, indicating cell death, although, at the same time they were all able to grow back in the presence of a reduced number of yeasts, suggesting a dose-dependent effect; and that (ii) enzymatic and siderophore activity was low/absent, indicating little influence in the mode of action. Importantly, we observed the existence of several antagonism-related features, such as coiled and emptied hyphae and yeasts cells inside the hyphae of C. godetiae and P. cinnnamomi. Results suggest that W. anomalus acts as a predatious yeast, using specific strategies according to its target, corroborating this yeast’s potential for field application in phytosanitary control.