This study mainly evaluated the broad-spectrum fungicidal activity of ε-poly L lysine (ε-PL) against 12 pathogenic fungi. We further demonstrated synergistic antifungal activity of ε-PL combined with wuyiencin against Botryris cinerea. The combined bio-fungicide achieved an inhibition rate of 100% for mycelial growth using ε-PL at 500 μg/mL + wuyiencin at 50 μg/mL and for spore germination using ε-PL at 200 μg/mL + wuyiencin at 80 μg/mL in vitro. This synergistic spore and mycelia-damaging effect of the combination was confirmed using scanning electron microscopy. In vivo assays with combined bio-fungicide (1500 μg/mL ε-PL + 60 μg/mL wuyiencin) on detached leaves showed depressed growth and development of the spores of B. cinerea. The synergistic effect was further tested in combinations of ε-PL with wuyiencin by measuring the fractional inhibition concentration index (FICI) value below 0.5. Moreover, ε-PL and wuyiencin inoculation before B. cinerea infection significantly increased the superoxide dismutase, peroxidase, catalase, and phenylalanine ammonia-lyase activities, which suggested their involvement in tomato defense responses to disease to minimize damage to B. cinerea. These findings revealed that a combined bio-fungicide comprising ε-PL and wuyiencin had a good prospect for controlling plant fungal disease.

Botrytis cinerea is a necrotrophic model fungal plant pathogen that causes grey mould, a devastating disease responsible for large losses in the agriculture sector. As important targets of fungicides, membrane proteins are hot spots in the research and development of fungicide products. We previously found that membrane protein Bcest may be closely related to the pathogenicity of Botrytis cinerea. Herein, we further explored its function. We generated and characterised ΔBcest deletion mutants of B. cinerea and constructed complemented strains. The ΔBcest deletion mutants exhibited reduced conidia germination and germ tube elongation. The functional activity of ΔBcest deletion mutants was investigated by reduced necrotic colonisation of B. cinerea on grapevine fruits and leaves. Targeted deletion of Bcest also blocked several phenotypic defects in aspects of mycelial growth, conidiation and virulence. All phenotypic defects were restored by targeted-gene complementation. The role of Bcest in pathogenicity was also supported by reverse-transcriptase real-time quantitative PCR results indicating that melanin synthesis gene Bcpks13 and virulence factor Bccdc14 were significantly downregulated in the early infection stage of the ΔBcest strain. Taken together, these results suggest that Bcest plays important roles in the regulation of various cellular processes in B. cinerea.