Inhibitory effect of Bacillus subtilis WL-2 and its IturinA lipopeptides against Phytophthora infestans

Potato late blight triggered by Phytophthora infestans ((Mont.) de Bary) represents a great food security threat worldwide and is difficult to control. Currently, Bacillus spp. have been considered biocontrol agents to control many fungal diseases. Here, Bacillus subtilis WL-2 was selected as the antifungal strain with the most potential against P. infestans mycelium growth. Additionally, the functional metabolites extracted from WL-2 were identified as IturinA-family cyclic lipopeptides (CLPs) via high-performance liquid chromatography (HPLC) and electrospray ionization mass spectrometry (ESI-MS). Analyses using scanning and transmission electron microscopy (SEM and TEM) revealed that IturinA caused a change in the mycelial surface and damage to the internal cell structure, including cell membrane disruption and irregular organelle formation. Moreover, propidium iodide staining and nucleic acid and protein release were detected to clarify the cell membrane damage caused by IturinA. Additionally, IturinA triggered reactive oxygen species (ROS) generation and malondialdehyde (MDA) production. Mitochondrial membrane potential (MMP), mitochondrial respiratory chain complexes activity (MRCCA), respiratory control rate (RCR), and oxidative phosphorylation efficiency (P/O) assays indicated that P. infestans mitochondria affected by IturinA were so seriously damaged that the MMP and MRCCA declined remarkably and that mitochondrial ATP production ability was weakened. Therefore, IturinA induces cell membrane damage, oxidative stress, and dysfunction of mitochondria, resulting in P. infestans hyphal cell death. As such, the results highlight that B. subtilis WL-2 and IturinA have great potential as candidates for inhibiting P. infestans mycelium growth and controlling potato late blight.\n\nIMPORTANCEPotato (Solanum tuberosum L.) is the fourth most common global food crop, and its planting area and yield increase yearly. Notably, in 2015, China initiated a potato staple food conversion strategy, and by 2020, approximately 50% of potatoes will be consumed as a staple food. The plant pathogen fungus Phytophthora infestans ((Mont.) de Bary) is the culprit of potato late blight; however, biological agents rather than chemicals are highly necessary to control this threatening disease. In this study, we discovered an antifungal substance, IturinA, a lipopeptide produced by Bacillus subtilis WL-2. Moreover, our research revealed the actual mechanism of IturinA against P. infestans mycelium growth and clarified the potential of B. subtilis WL-2 and IturinA as a biocontrol agent against P. infestans mycelium growth as well as for controlling the development of late blight in potato cultivation.