Evaluation of the biocontrol activity of Frateuria defendens-derived metabolites against mollicutes

Frateuria defendens is a candidate biocontrol agent that has been shown to reduce phytoplasma-related disease symptoms in grapevines and periwinkle plants. While a crude filtrate prepared from F. defendens can inhibit mollicute growth, the specific growth parameters for this bacterium, necessary to enhance this protective inhibitory response, remain unknown. Moreover, the separation of filtrate preparations from bacterial cells via centrifugation and filtration is laborious and time-consuming. As such, the present study was conducted to define the optimal growth conditions associated with maximal inhibitory activity of F. defendens and to establish a better approach to separating these bacterial cells from their secreted metabolites. To conduct these analyses, F. defendens was cultured in a range of media types, while associated inhibitory effects were tested in vitro using Spiroplasma melliferum as a model mollicute bacterium, and in planta using phytoplasma-infected periwinkle plantlets. These analyses revealed F. defendens growth patterns change based upon media composition, with filtrates prepared from a specific rich medium (S-medium) exhibiting beneficial activities, including the inhibition of S. melliferum and enhanced plant growth. When F. defendens cells were grown within semi-permeable, membrane-coated Small Bioreactor Platform (SBP) capsules, they could be more readily separated from the secreted metabolite fraction, obviating the need for filtration and/or centrifugation. This study is the first to have reported the use of SBP capsules to separate bacterial cells from their secreted metabolites under sterile conditions while retaining the ability of these metabolites to inhibit S. melliferum growth and to benefit the host plant. The results highlight promising new approaches to the effective biocontrol of phytoplasma-driven diseases in grapevines and other economically important plant species.