Study On The Anti-Fungal Mechanism Of Bacillus Amyloliquefaciens WH1

Some species of Bacillus could produce lipopeptides for controlling plant pathogenic fungi. In this study, we isolated a strain of B. amyloliquefaciens, called as WH1, from the rice root. WH1 could produce lipopeptide, WHlfungin, to inhibit several plant pathogenic fungi. WH1fungin could be isolated from the culture of WH1 by extraction and gel filtration, and characterized as an anti-fungal lipopeptide (surfactin) by thin layer chromatography, infrared spectrum, Tricine-SDS-PAGE and mass spectrum. We also studied on the anti-fungal mechanism of it.After treated by WH1fungin, it was found that WH1fungin could directly kill some fungal cells by eliciting pores on membrane, while WHlfungin also could induce apoptosis. Classic apoptotic markers such as nucleus condensation, ROS accumulation, PS externalization, DNA strand breaks and high caspase-like activity were detectable.Because ROS is a key factor for fungal apoptosis, further investigation was done for elucidating the role of ROS in apoptotic fungal cells. When fungal cells were treated by WHlfungin, antioxidants such as ascorbic acid, proline and reduced glutathione (GSH) were used for cleaning ROS respectively. It was found that ascorbic acid had no effect on the ROS, while proline and GSH could significantly inhibit ROS accumulation in WHlfungin-treated fungal cells. Unfortunately, clean of ROS by proline and GSH couldn't inhibit the relevant PS externalization in fungal cells.It was reported that several cyclic lipopeptides could inhibit the activity of fungal glucane synthase, resulting in a synthesis decrease of callose. New generated fungal cells without enough callose for forming cell wall were easily found in apoptosis. In this study we found the activity of fungal glucane synthase was obviously decreased, and correspondent apoptotic markers could be detected in WHlfungin-treated fungal cells.In order to detect the targeted proteins of WH1fungin, we used WH1fungin conjugated Sepharose 4B to isolate proteins in fungal cells. A purified protein was collected after isolation by this affinity chromatography, and was identified as a mitochondrial F-ATPase by peptide mass fingerprinting. After treated by FITC labeled WH1fungin, it was showed that WH1fungin could bind to the mitochondrial memebrane of fungal cells. After treated by WH1fungin, ATPase activity was significantly lower than control groups. It's known that oligomycin is an inhibitor of mitochondrial FoF1-ATPase of fungal cells. In this study more ROS and PS externalization was observed in fungal cells when oligomycin was used for treating fungal cells together with WHlfungin. All of these data verified that WH1fungin could interact with ATPase on the mitochondrial membraneAs a conclusion, low level of WH1fungin produced by WH1 could suppress the fungal glucane synthase, inhibit the mitochondrial ATPase, and then induce apoptosis in fungal cells. Normally Bacillus couldn't produce enough lipopeptide to elicit pores on fungal membrane in the natural environment, so we deduced that induction of apoptosis might be the common anti-fungal mechanism of Bacillus in the natural habitat.