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.

The Characterization and the Biological Activity of Phytotoxin Produced by Paraphoma radicina.

Paraphoma radicina is a new pathogen that causes alfalfa paraphoma root rot (APRR), leading to alfalfa production losses. The resistance levels of 30 alfalfa cultivars to APRR have already been characterized. However, the pathogenic mechanism of P. radicina is still unclear. This study aimed to assess the effects of a crude toxin extracted from P. radicina cell-free culture filtrate (CFCF) on susceptible and resistant cultivars of alfalfa. Meanwhile, the crude toxin components were detected using gas chromatography-mass spectrometry (GC-MS) analysis. CFCF cultured in MEB medium for 14 days and crude toxin extracted by ethyl acetate induced significant phytotoxicity caused the average lesion areas of 5.8 and 3.9 mm2, respectively, on alfalfa leaves. The crude toxin exhibited resistance to high temperature, as shown by a lesion area of 3.6 mm2 when treated at 120 °C for 30 min. Different concentrations of the crude toxin in water and MS medium had different effects on susceptible and resistant cultivars. Moreover, the crude toxin affected the plasma membrane, mitochondria, and nuclear membranes of alfalfa root cortical cells. Further, it induced significant phytotoxicity on Sonchus oleraceus L., Capsella bursa-pastoris (Linn.) Medic, and Chenopodium album L. Agropyron cristatum L. (average lesion areas; 11.6, 15.8, 21.4, and 6.2 mm2, respectively), indicating that the crude toxin of P. radicina is a non-host-selective toxin. GC-MS analysis detected four possible active substances in the toxin (3-hydroxypyridine, 5-methylresorcinol, 3-Hydroxypropionic acid, and 4-Hydroxyphenylethanol). Therefore, this study may provide insight into the pathogenic mechanism of P. radicina to alfalfa.