Biocontrol Potential of Bacillus amyloliquefaciens LYZ69 Against Anthracnose of Alfalfa (Medicago sativa)

Anthracnose is a destructive disease of alfalfa (Medicago sativa) that causes severe yield losses. Biological control can be an effective and eco-friendly approach to control this alfalfa disease. In the present study, Bacillus amyloliquefaciens LYZ69, previously isolated from healthy alfalfa roots, showed a strong in vitro antifungal activity against Colletotrichum truncatum, an important causal agent of anthracnose of alfalfa. The strain LYZ69 protected alfalfa plants (biocontrol efficacy of 82.59%) from anthracnose under greenhouse conditions. The cell-free culture (CFC) of LYZ69 (20%, vol/vol) caused 60 and 100% inhibition of mycelial growth and conidial germination, respectively. High-performance liquid chromatography tandem mass spectrometry separated and identified cyclic lipopeptides (LPs) such as bacillomycin D and fengycin in the CFC of LYZ69. Light microscopy and scanning electron microscopy revealed that the mixture of cyclic LPs produced by LYZ69 caused drastic changes in mycelial morphology. Fluorescence microscopy showed that the LPs induced reactive oxygen species accumulation and caused apoptosis-like cell death in C. truncatum hyphae. In summary, our findings provide evidence to support B. amyloliquefaciens LYZ69 as a promising candidate for the biological control of anthracnose in alfalfa.