解淀粉芽孢杆菌B15抑菌物质对葡萄灰霉病灰葡萄孢的抑菌机理

Inhibition of antifungal substances from Bacillus amyloquefaciens B15 against Botrytis Cinerea—the agent of “gray mold” of grape

摘要:

[目的]利用荧光显微镜和激光共聚焦扫描显微镜技术初步探讨解淀粉芽孢杆菌（Bacillus amyloquefaciens）B15菌株发酵液中的抑菌混合物质伊枯草菌素A（iturin A）和芬芥素（fengycin）对葡萄灰霉病病原菌灰葡萄孢（Botrytis cinerea）的抑菌机理。[方法]采用琼脂稀释法讨论解淀粉芽孢杆菌B15发酵液对灰葡萄孢的抑菌活性。利用台盼蓝（trypan blue）染色、4'，6-二脒基-2-苯基吲哚（DAPI）、双氢罗丹明123（DHR123）、钙离子探针fluo-3/am和Annexin V-PI探针染色来观察解淀粉芽孢杆菌B15发酵液对灰葡萄孢细胞膜和菌丝形态、细胞核、活性氧、钙离子和磷脂酰丝氨酸层的影响。[结果]抑菌活性实验发现解淀粉芽孢杆菌B15发酵液对灰葡萄孢具有良好抑菌效果。荧光显微镜台盼蓝染色观察发现，经B15发酵液处理过的灰葡萄孢出现菌丝畸形、菌丝体粗大、尖端肿胀并被染成蓝色和明显的液泡化现象。同时未在处理组中观察到细胞内容物泄漏，说明处理组菌丝细胞膜未发生破损。该结果表明在此次试验中，B15发酵液中的抑菌有效物质不以破损细胞膜的方式直接导致灰葡萄孢的死亡。激光共聚焦显微镜观察结果发现，处理组的灰葡萄孢菌丝出现典型的细胞凋亡现象、染色质固缩、细胞核裂解、磷脂酰丝氨酸层外翻、活性氧和钙离子积累。[结论]该实验表明解淀粉芽孢杆菌B15发酵液以诱导细胞凋亡的形式来抑制灰葡萄孢菌丝的生长。

Abstract:

[Objective] The present study aimed to investigate the inhibition of cyclic lipopeptides (iturin A and fengycin) produced by Bacillus amyloquefaciens B15 against Botrytis cinerea by using laser confocal scanning microscopy and fluorescence microscopy. [Methods] The agar diffusion method was applied to investigate the antifungal activity of B15 strain against Botrytis cinerea. A 7-mm agar plug of Botrytis cinerea was placed on the PDA plate containing B15 culture supernatant at the concentration of 1:20. The hyphal viability was determined by staining the hyphae of Botrytis cinerea with trypan blue. Then the classical cell apoptotic assay was applied by staining cell nucleus, reactive oxygen species, calcium ions, and phosphatidylserine of hyphae with DAPI, Dihydrorhodamine 123, fluo-3/am and Annexin V-PI probe. [Results] The antagonistic assay showed that the B15 strain displayed evident antifungal activity against Botrytis cinerea. From the trypan blue assay, it was evident to observe the distorted hyphal structure of Botrytis cinerea in treated group, including conglobation of branch tips, swollen hyphae, hyphal vacuolation, and shrinkage under fluorescence microscopy. The laser confocal scanning microscopy observation of Botrytis cinerea showed that the typical cell apoptosis pattern appeared in the treated group, including the nuclear fragmentation and condensation, accumulation in reactive oxygen species and Ca²⁺, and the flip-flop of phosphatidylserine. [Conclusion] The combined results of the trypan blue assay and cell apoptotic assay indicated that B15 culture supernatant inhibited the growth of Botrytis cinerea in the way of inducing apoptotic-like cell death in Botrytis cinerea, instead of damaging the cell membrane and directly contributing to the death of mycelium.