FliZ调控枯草芽孢杆菌Bs916生物膜形成 及其对水稻纹枯病的防治效果

doi:10.3864/j.issn.0578-1752.2020.01.005

摘要/Abstract

摘要：

【目的】发掘并鉴定枯草芽孢杆菌（Bacillus subtilis）Bs916生物膜形成调控新基因,检测其对Bs916生物膜形成能力和对水稻纹枯病防治效果的影响。【方法】利用基因同源重组技术构建fliZ基因位点的单敲除突变株,通过干重分析法来验证其生物膜形成的缺陷;利用平板对峙试验检测fliZ突变株和Bs916对水稻纹枯病菌（Rhizoctonia solani）的抑菌效果;利用高效液相色谱（HPLC）检测fliZ突变株和Bs916中影响防治效果的3种脂肽类抗生素表面活性素、杆菌霉素L和泛革素的相对产量;利用绿色荧光标记技术构建Bs916与fliZ突变株的GFP标记菌株,观察两者在水稻茎秆定殖能力变化;检测fliZ突变株和Bs916对水稻纹枯病的防治效果。【结果】成功构建了fliZ位点单敲除突变株,与对照组Bs916的三维立体结构生物膜相比仅能形成平面二维结构生物膜,呈现破碎状态,证明其生物膜形成存在显著缺陷;对生物膜干重进行定量分析发现fliZ突变株生物膜干重仅为对照组Bs916的23%,进一步验证了fliZ突变株生物膜形成能力显著下降;游动性试验发现fliZ突变株菌体扩展直径仅为Bs916的32%,证明fliZ突变株的游动能力显著下降;抑菌试验显示两者抑菌带宽基本一致,证明fliZ突变株对水稻纹枯病菌的抑菌能力与Bs916相比无显著差异;成功检测了fliZ突变株和Bs916合成的3种脂肽类抗生素表面活性素、杆菌霉素L和泛革素的相对产量,fliZ突变株中杆菌霉素L相对产量显著增加1倍,而表面活性素和泛革素相对产量与Bs916相比无显著差异;水稻茎秆定殖试验发现fliZ突变株菌体数量显著低于Bs916,在水稻纹枯病病斑附近不出现显著的聚集效应,呈现无序分布状态,证明fliZ突变株与Bs916相比在水稻茎秆上的定殖能力显著下降;对水稻纹枯病的田间防治效果试验显示,fliZ突变株第6—15天防治效果介于6.0%—20.7%,显著低于Bs916的36.0%—57.6%,证明fliZ突变株对水稻纹枯病防治效果显著下降。【结论】鉴定的Bs916生物膜新调控基因fliZ位于控制鞭毛运动的信号通路,直接作用于菌体的游动与扩张,显著单一调控生物膜形成与对水稻纹枯病的防治效果。

关键词: 枯草芽孢杆菌Bs916, 调控基因, 生物膜, 脂肽抗生素, 定殖, 水稻纹枯病, 防治效果

Abstract:

【Objective】The objective of this study is to discover and identify new regulatory genes on biofilm formation of Bacillus subtilis Bs916, detect its effect on biofilm formation of Bs916 and biocontrol efficacy on rice sheath blight.【Method】The single knockout mutant of Bs916 at fliZ was construct by homologous recombination, and its defects in biofilm formation were verified by dry weight analysis. The anti-bacterial effect of fliZ mutant and Bs916 on rice sheath blight pathogen (Rhizoctonia solani) was detected by flat panel. The relative production of 3 lipopeptide antibiotics (LPs) surfactin, bacillomycin L, and fengycin in fliZ mutant and Bs916 was detected by HPLC. The GFP-labeled strains of Bs916 and fliZ mutant were constructed by green fluorescent labeling, the colonization ability of them in rice stalks was observed, and the biocontrol efficacy of fliZ mutant and Bs916 on rice sheath blight was detected.【Result】The single knockout mutant of Bs916 at fliZ was successfully constructed. Compared with the three-dimensional structure biofilm of the control group Bs916, fliZ mutant only formed a planar two-dimensional structure biofilm, appeared broken form, which proved that it had significant defects in biofilm formation. Quantitative analysis of the dry weight of biofilms showed that the biofilm dry weight of fliZ mutant was only 23% of the control group Bs916, which further verified that the biofilm formation ability of fliZ mutant was significantly decreased. The motility test found that the expanded diameter of fliZ mutant was only 32% of Bs916, which proved that the swimming ability of fliZ mutant was significantly reduced. The bacteriostatic test showed that the antibacterial bandwidth of the two strains was basically the same, and it is proved that the antibacterial activity of fliZ mutant against R. solani was not significantly different from that of Bs916. The relative production of three LPs bacillomycin L, surfactin, and fengycin in fliZ mutant and Bs916 was successfully detected. Compared with Bs916, the relative production of bacillomycin L was significantly increased by 1 time in fliZ mutant, but the relative production of surfactin and fengycin was not significantly different from that of Bs916. The colonization test of rice stalks showed that the number of fliZ mutant was significantly lower than that of Bs916, and there was no significant aggregation effect near the rice sheath blight lesions, and presented an unordered state, which proved that the colonization ability of fliZ mutant on rice stalks was significantly lower than that of Bs916. The field biocontrol trials against rice sheath blight showed that biocontrol efficacy of fliZ mutant ranged from 6.0% to 20.7% on days 6-15, which was significantly lower than that of Bs916 (36.0%-57.6%). It was proved that the biocontrol efficacy of fliZ mutant on rice sheath blight was significantly reduced.【Conclusion】The new regulatory gene fliZ of Bs916 biofilm identified in this study is located in the signal pathway controlling flagellar movement, directly acts on swimming and expansion of the bacteria, and can significantly control the biofilm formation and its biocontrol efficacy on rice sheath blight.

Key words: Bacillus subtilis Bs916, regulated genes, biofilm, lipopeptide antibiotics (LPs), colonization, rice sheath blight, biocontrol efficacy

周华飞,杨红福,姚克兵,庄义庆,束兆林,陈志谊. FliZ调控枯草芽孢杆菌Bs916生物膜形成及其对水稻纹枯病的防治效果[J]. 中国农业科学, 2020, 53(1): 55-64.

HuaFei ZHOU,HongFu YANG,KeBing YAO,YiQing ZHUANG,ZhaoLin SHU,ZhiYi CHEN. FliZ Regulated the Biofilm Formation of Bacillus subtilis Bs916 and Its Biocontrol Efficacy on Rice Sheath Blight[J]. Scientia Agricultura Sinica, 2020, 53(1): 55-64.

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引物Primer	序列Sequence (5′-3′)	来源Source
SpecF	TTTGGATCCCTGCAGCCCTGGCGAATG	本试验This study
SpecR	TTTGAATTCAGATCCCCCTATGCAAGG	本试验This study
FliZF	TTTAAGCTTTACATCCGTTCCCTGCTTTT	本试验This study
FliZR	TTTGGATCCGGCCTTTCTTCTTTCCTTCA	本试验This study

菌株Strain	6 d	9 d	12 d	15 d
Bs916	57.6a	51.2a	43.0a	36.0a
ΔfliZ	20.7b	11.9b	7.3b	6.0b