周华飞^1,2, 杨红福¹, 姚克兵¹, 庄义庆¹, 束兆林¹, 陈志谊^,31 江苏丘陵地区镇江农业科学研究所,江苏句容 212400
2 南京农业大学植物保护学院,南京 210095
3 江苏省农业科学院植物保护研究所, 南京210014

FliZ Regulated the Biofilm Formation of Bacillus subtilis Bs916 and Its Biocontrol Efficacy on Rice Sheath Blight

ZHOU HuaFei^1,2, YANG HongFu¹, YAO KeBing¹, ZHUANG YiQing¹, SHU ZhaoLin¹, CHEN ZhiYi^,3 1 Zhenjiang Institute of Agricultural Sciences in Hilly Region of Jiangsu, Jurong 212400, Jiangsu
2 College of Plant Protection, Nanjing Agricultural University, Nanjing 210095
3 Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, Nanjing 210014

通讯作者: 陈志谊,Tel：025-84390393;E-mail：njaujaas@163.com

责任编辑: 岳梅
收稿日期:2019-07-1接受日期:2019-08-4网络出版日期:2020-01-01

基金资助:

国家重点研发计划.2017YFD0201100 江苏省农业科技自主创新资金.CX173023 镇江市农业科学院青年基金.QNJJ2017006

Received:2019-07-1Accepted:2019-08-4Online:2020-01-01
作者简介 About authors
周华飞,Tel：0511-80978079;E-mail：zhhf2010@126.com。








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.
Keywords：Bacillus subtilis Bs916;regulated genes;biofilm;lipopeptide antibiotics (LPs);colonization;rice sheath blight;biocontrol efficacy


PDF (1747KB)元数据多维度评价相关文章导出EndNote|Ris|Bibtex收藏本文
本文引用格式
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 doi:10.3864/j.issn.0578-1752.2020.01.005
ZHOU HuaFei, YANG HongFu, YAO KeBing, ZHUANG YiQing, SHU ZhaoLin, CHEN ZhiYi. FliZ Regulated the Biofilm Formation of Bacillus subtilis Bs916 and Its Biocontrol Efficacy on Rice Sheath Blight[J]. Scientia Acricultura Sinica, 2020, 53(1): 55-64 doi:10.3864/j.issn.0578-1752.2020.01.005

0 引言

【研究意义】由立枯丝核菌（Rhizoctonia solani）引起的水稻纹枯病（rice sheath blight）已是影响稻米产量和品质的三大主要病害之首^[1,2,3],水稻不同生长时期均会受到该病危害,在我国长江流域地区发生尤为严重,降低水稻千粒重和产量,普遍减产15%—20%,严重减产最高可达50%^[4]。选育抗病品种和使用药剂是防治水稻纹枯病最有效的两种手段。以芽孢杆菌（Bacillus）为代表的微生物农药在防治水稻纹枯病领域已有长足发展,同时防病抗病机制也逐步清晰^[5,6,7]。除了产生可直接抑制病原菌扩展的抗生素之外,生物膜（biofilm）是影响芽孢杆菌等微生物菌株在寄主表面定殖的关键因子,进而直接影响到对病害的防治效果^[5,8-9]。芽孢杆菌生物膜基本构成组分和骨架已十分清晰^{[10,11,12,13]},但是其复杂的调控信号通路目前尚未形成框架图^[14]。因此,进一步挖掘和鉴定芽孢杆菌生物膜调控新基因,完善生物膜调控网络,对应用微生物菌株防治植物病害具有重要意义。【前人研究进展】芽孢杆菌鞭毛形成主要由一级主调控操纵子flhDC及其控制的二级fli调控通路来完成其运动功能,已有报道揭示该二级fli通路又与生物膜形成与调控具有十分紧密的联系,但具体作用机制不明确。BISCHOFF等^[15]报道在枯草芽孢杆菌（Bacillus subtilis）中缺失表达FliZ蛋白不能形成完整的鞭毛控制单元,因而显著降低其游动性;XU等^[16]研究发现,假结核耶尔森菌（Yersinia pseudotuberculosis）中缺失表达FliS蛋白显著降低鞭毛的长度与菌体的游动能力,互补菌株则能够恢复鞭毛正常的游动能力;LUO等^[5]研究发现,在Bs916中缺失表达脂肽抗生素杆菌霉素L（bacillomycin L）和表面活性素（surfactin）显著降低菌体游动能力,同时显著降低了生物膜形成强度,通过体外物质回补方式也能够恢复菌体游动性与生物膜形成能力;杨丙烨等^[17]研究表明,在西瓜细菌性果斑病菌（Acidovorax citrulli）中缺失表达FliS蛋白导致菌体游动性与菌膜（生物膜）形成和病原菌对西瓜果实致病能力均显著下降,回补菌株则基本能够恢复上述表型性状,说明鞭毛介导的菌体游动能力与生物膜形成呈现正相关。生物膜的主要构成组分是蛋白质TasA、胞外多糖EPS及其他一些生物膜表层蛋白BslA（YuaB）等^{[10-13,18-21]}。芽孢杆菌生物膜基因调控网络极其复杂,目前报道的主要有Spo0A-KinA-E、SinR/SinI/SlrR、AbbA/AbrB和DegU/DegS等途径,均为通过调控epsA-O和tapA-sipW-tasA操纵子的启动子来控制生物膜形成的强弱。SinR可以分别与epsA-O和tapA-sipW-tasA操纵子结合来抑制生物膜基本组分的转录合成,进而严重降低生物膜形成强度,SinI则能够解除该种效应^[22,23,24]。AbrB结合在tapA-sipW-tasA操纵子启动区-133和-182位来实现抑制其转录表达,进而减弱生物膜形成强度^{[25,26,27,28]}。Spo0A直接通过其磷酸化水平决定形成生物膜或芽孢,通过抑制负调控因子AbrB来增强生物膜的形成^{[25-26,29-32]}。【本研究切入点】笔者实验室前期基础研究以Bs916转座子标签库为出发点,以生物膜形成缺陷为筛选表型进行筛库试验,获得具有生物膜形成缺陷的fliZ突变株,进行生物信息学分析获取生物膜调控新基因fliZ（Gene ID：16551915）,该基因位于芽孢杆菌控制鞭毛运动的信号通路中,被预测为鞭毛调控蛋白。【拟解决的关键问题】采用定向敲除突变位点基因的方法验证生物膜形成缺陷,检测突变株脂肽类抗生素产量、对水稻纹枯病菌的抑菌能力及在水稻茎秆的定殖能力,探讨该调控基因对生物膜形成和对水稻纹枯病防治效果的重要作用。

1 材料与方法

试验于2016—2018年在江苏丘陵地区镇江农业科学研究所和江苏省农业科学院植物保护研究所完成。

1.1 材料

1.1.1 细菌、病原菌及其生长条件

枯草芽孢杆菌Bs916由江苏省农业科学院植物保护研究所生防与稻病研究室提供,水稻纹枯病菌为江苏丘陵地区镇江农业科学研究所植保研究室分离纯化保存菌株。Bs916培养基为LB（Luria broth）培养基：10 g·L^-1胰蛋白胨,5 g·L^-1酵母提取物,10 g·L^-1 NaCl^[14,33-34];水稻纹枯病菌培养基为PDA培养基：马铃薯块200 g·L^-1,葡萄糖20 g·L^-1,琼脂20 g·L^-1[14,34];生物膜诱导培养基为MSgg培养基：5 mmol·L^-1磷酸钾（pH 7）,100 mmol·L^-1 Mops（pH 7）,2 mmol·L^-1氯化镁,700 μmol·L^-1氯化钙,50 μmol·L^-1氯化锰,50 μmol·L^-1氯化铁,1 μmol·L^-1氯化锌,2 μmol·L^-1维生素B1,0.5%甘油,0.5%谷氨酸,50 μg·mL^-1色氨酸,50 μg·mL^-1苯丙氨酸^[14,20]。

1.1.2 供试引物

本试验引物合成和序列测序均由上海生工生物工程有限公司完成,所用引物如表1所示。

Table 1
表1
表1本试验所用引物
Table 1Primers used in this study

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

新窗口打开|下载CSV

1.2 fliZ目标基因的获取、定向敲除突变株的构建、GFP标记与生物膜表型缺陷的验证

在实验室前期工作基础上（包含部分未发表数据^[34]）通过构建转座子随机插入突变体库,以生物膜形成表型缺陷作为筛库标准,筛选到fliZ突变株,经Southern blot拷贝数检测和PCR双重验证后确认单插入位点位于fliZ。从质粒pDG1728上扩增（SpecF,SpecR）得到由壮观霉素启动子和合成基因组成的基因盒,经BamHI和EcoRI酶切装载入质粒pUC19构成新质粒pUCSpec。从Bs916全基因组PCR扩增（FliZF,FliZR）fliZ部分片段（582 bp）,片段纯化经BamHI和HindIII双切后装载入质粒pUCSpec构成新质粒pUCSpec-fliZ。质粒pUCSpec-fliZ经感受态转化方式转化进入Bs916野生型菌株内部,发生单交换-同源重组突变,在含有壮观霉素的固体LB培养基中定向选取突变株并进行PCR验证,构建fliZ定向敲除突变株ΔfliZ。

质粒pRp22-gfp（江苏省农业科学院植物保护研究所保存）分别经感受态转化进入突变株ΔfliZ和野生型菌株Bs916构成携带绿色荧光标记的突变株ΔfliZ-GFP和Bs916-GFP,用于定殖能力检测。突变株ΔfliZ单菌落接种于含有100 µg·mL^-1壮观霉素的LB液体培养基37℃条件下200 r/min过夜培养12 h。4 mL含有终浓度20 µg·mL^-1刚果红和10 µg·mL^-1考马斯亮蓝MSgg液体培养基加入12孔板中,上述过夜培养新鲜菌液200 µL加入上述MSgg培养基中诱导产生生物膜,分批次于2 mL离心管中离心去上清液倒置空干,37℃条件下烘干称重分析^[35]。每个处理重复3次。

1.3 ΔfliZ突变株和Bs916游动能力检测

检测ΔfliZ突变株和Bs916在LB游动性培养基上菌落直径。在包含20 µg·mL^-1刚果红,10 µg·mL^-1考马斯亮蓝和0.7%琼脂粉的培养基上分别滴加1 µL过夜培养新鲜的Bs916和ΔfliZ菌液,30℃条件下倒置培养24 h,分别测量菌体直径,3次重复。

1.4 ΔfliZ突变株和Bs916对水稻纹枯病菌抑菌能力与3种脂肽抗生素分泌量的检测

挑取ΔfliZ突变株单菌落接种至含有100 µg·mL^-1壮观霉素LB液体培养基,Bs916单菌落接种至不含任何抗生素的LB培养基中,均于37℃过夜培养。分别取2 µL过夜培养新鲜菌液滴加在中央已接种水稻纹枯病菌菌饼的培养皿中,28℃条件静置培养24—72 h,分别测量Bs916和ΔfliZ突变株抑菌圈直径,每个处理重复3次。

分别接种上述过夜培养新鲜的ΔfliZ突变株和Bs916菌液10 µL于50 mL不含抗生素LB培养基的三角瓶中,200 r/min转速37℃条件下摇培48 h,10 000 r/min转速离心20 min分别获取上清液,使用HCl调节pH到2.0进行静置沉淀3 h,12 000 r/min转速离心30 min获取沉淀,自然干燥之后沉淀分别溶解于2 mL甲醇溶液,经0.22 µm滤膜过滤去除杂质用于高效液相色谱（HPLC）上样检测。采用安捷伦1200系HPLC和C18色谱柱（5 μm,4 mm×250 mm,Frankfurt,Germany）进行分离3种脂肽类抗生素杆菌霉素L、表面活性素和泛革素（fengycin）的相对产量,流动相及检测条件分别为杆菌霉素L（乙腈﹕水﹕三氟乙酸=40﹕60﹕0.5,V/V/V）,表面活性素（乙腈﹕水﹕三氟乙酸=20﹕80﹕0.5,V/V/V）和泛革素（乙腈﹕水﹕三氟乙酸=50﹕50﹕0.5,V/V/V）,检测波长均为210 nm,流速为0.8 mL·min^-1,柱温为30℃。每个处理重复3次。

1.5 ΔfliZ突变株和Bs916在水稻茎秆定殖能力和对水稻纹枯病防治效果的测定

挑取ΔfliZ-GFP突变株单菌落接种至含有100µg·mL^-1壮观霉素LB液体培养基,Bs916-GFP单菌落接种至不含有抗生素的LB液体培养基,37℃条件过夜培养。分别取上述过夜培养的新鲜菌液10 µL于50 mL不含抗生素LB培养基的三角瓶中,200 r/min转速37℃条件下摇培24 h,分别喷施上述扩培菌液20 mL于已接种水稻纹枯病菌的水稻茎秆部位,不同时间段内取水稻茎秆样品于激光共聚焦显微镜下观察菌落的定殖情况。每个处理重复3次。

相同的处理方式分别处理ΔfliZ突变株和Bs916,分别将扩培之后20 mL新鲜菌液喷施于已接种水稻纹枯病菌的水稻茎秆部位,空白对照组CK仅接种水稻纹枯病菌,待对照组水稻纹枯病发病明显时测量病斑直径,分别计算对水稻纹枯病的防治效果。每个处理重复3次。

2 结果

2.1 ΔfliZ突变株生物膜形成表型缺陷的验证

采用定向突变的方式敲除了Bs916基因组中的fliZ位点序列,经MSgg培养基诱导检测ΔfliZ突变株形成的缺陷,结果如图1所示。对照野生型菌株Bs916能够形成具有三维结构的完整生物膜,轮廓清晰。ΔfliZ突变株也能够形成部分的生物膜组织,仅具有平面二维结构,呈现漂浮破碎状,分布均匀。干重分析显示,ΔfliZ突变株生物膜干重仅为Bs916的23%,生物膜形成能力显著下降。

图1

新窗口打开|下载原图ZIP|生成PPT
图1ΔfliZ突变株生物膜形成缺陷的验证

*: P<0.05
Fig. 1Verification of biofilm formation defects of ΔfliZ mutant

2.2 ΔfliZ突变株和Bs916游动能力

检测了对照野生型菌株Bs916和ΔfliZ突变株在菌体游动性表型上的差异,结果如图2所示,Bs916菌体在24 h铺满整个培养皿,ΔfliZ突变株在24 h与12—18 h相比无显著差异,与Bs916相比具有显著差异,扩展直径仅为Bs916的32%。

图2

新窗口打开|下载原图ZIP|生成PPT
图2ΔfliZ突变株和Bs916游动能力测定

Fig. 2Swimming detection of ΔfliZ mutant and Bs916

2.3 ΔfliZ突变株和Bs916对水稻纹枯病菌的抑菌能力

野生型菌株Bs916对水稻纹枯病抑菌带宽为3.2 mm,ΔfliZ突变株抑菌带宽是3.3 mm,Bs916和ΔfliZ突变株对水稻纹枯病菌均具有正常的抑菌能力,两者无显著差异（图3）。

图3

新窗口打开|下载原图ZIP|生成PPT
图3ΔfliZ突变株和Bs916抑菌能力测定

Fig. 3Antibacterial ability detection of ΔfliZ mutant and Bs916

2.4 ΔfliZ突变株和Bs916分泌的3种脂肽抗生素的相对产量

利用HPLC检测了同等发酵条件下野生型菌株Bs916和ΔfliZ突变株产杆菌霉素L、表面活性素和泛革素的相对产量,如图4所示,Bs916分泌的杆菌霉素L、表面活性素和泛革素经HPLC检测分别存在3、6和5个峰,分子量数据定性由本实验室前期数据确定^[5,36]。结果显示杆菌霉素L相对产量具有显著差异,ΔfliZ突变株相对产量是Bs916的两倍,而表面活性素和泛革素两者无显著差异。

图4

新窗口打开|下载原图ZIP|生成PPT
图4ΔfliZ突变株和Bs916产3种脂肽抗生素相对产量

A—C图蓝色代表Bs916,红色代表ΔfliZ突变株 In fig A-C, blue represents Bs916, red represents ΔfliZ mutant. A：杆菌霉素L 相对产量Relative production of bacillomycin L;B：表面活性素相对产量Relative production of surfactin;C：泛革素相对产量Relative production of fengycin;D：3种脂肽抗生素相对产量柱形图Histogram of relative production of 3 lipopeptide antibiotics. *: P<0.05
Fig. 4Relative production of 3 lipopeptide antibiotics of ΔfliZ mutant and Bs916

2.5 ΔfliZ突变株和Bs916在水稻茎秆的定殖能力

成功构建了ΔfliZ突变株和Bs916分别携带绿色荧光标记的菌株ΔfliZ-GFP和Bs916-GFP。利用绿色荧光蛋白表达技术,在激光共聚焦显微镜下成功观测到ΔfliZ-GFP和Bs916-GFP在水稻茎秆上定殖能力的差异（图5）,水稻纹枯病菌从第3天显示发病迹象,ΔfliZ-GFP和Bs916-GFP菌体数量处于指数繁殖阶段,无明显的聚集效应。在第6天开始,在水稻纹枯病菌侵染部位Bs916菌群出现部分的聚集效应,ΔfliZ-GFP则无此种聚集效应。至第9天观测效果显著,Bs916菌体量增加显著,高度聚集在侵染病斑周围,呈片状分布,ΔfliZ-GFP菌体数量增加,但呈现游离状态分布。第12—15天由于病原菌在竞争中占据优势地位,菌体数量明显下降,Bs916仍具有聚集效应,但ΔfliZ-GFP菌体一直延续游离状态。

图5

新窗口打开|下载原图ZIP|生成PPT
图5ΔfliZ突变株和Bs916在水稻茎秆定殖能力观测

Fig. 5Observation on the colonization ability of rice stalk of ΔfliZ mutant and Bs916

2.6 ΔfliZ突变株和Bs916对水稻纹枯病的田间防治效果

野生型菌株Bs916防治效果由于水稻纹枯病菌的持续扩展侵染呈现下降趋势,ΔfliZ突变株同样呈现下降趋势,但显著低于Bs916的防治效果,说明该位点的突变显著降低了Bs916对水稻纹枯病的防治效果（表2）。

Table 2
表2
表2ΔfliZ突变株和Bs916对水稻纹枯病的防治效果
Table 2Biocontrol efficacy on rice sheath blight of ΔfliZ mutant and Bs916 (%)

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

同列数据后含有不同小写字母表示差异显著（P<0.05）
Different lowercases after the data indicate significant difference (P<0.05)

新窗口打开|下载CSV

3 讨论

水稻纹枯病是影响全球水稻主产区稻米产量的主要病害之一,在我国长江流域以南水稻种植地区广泛分布^[37,38]。防治水稻纹枯病主要依靠抗病品种选育和化学农药,但化学农药存在残留、抗药性产生和影响环境等问题,使得以芽孢杆菌为代表的绿色微生物农药得到长足发展。除直接分泌抗生素杀死或抑制病原真菌侵染和扩展外^[39],芽孢杆菌防治水稻纹枯病的前提是以生物膜的形式在水稻植株上成功定殖和繁殖。BIANCIOTTO等^[40]研究发现,强壮的生物膜能够增强荧光假单胞菌（Pseudomonas fluorescens）CHA0在胡萝卜根部的定殖能力,进而增强对病原菌侵入的抑制作用;BAIS等^[41]研究发现,枯草芽孢杆菌6051能够形成强大的三维结构生物膜定殖于拟南芥根部,抑制病原菌对拟南芥根部的侵染;TIMMUSK等^[42]研究发现,根际促生菌多粘类芽孢杆菌（Paenibacillus polymyxa）通过强大的生物膜结构定殖于植物根际并抵抗病原菌入侵;CHEN等^[43]发现并鉴定了6株生防菌,在培养基和番茄根部均能形成良好的生物膜结构以抑制番茄青枯病菌（Pseudomonas solanacearum）,进一步发现很多生物膜胞外聚合物基因得到增强表达。

本研究出发菌株Bs916是微生物农药“纹曲宁”的主效成分,被用于防治水稻纹枯病已长达15年,是一株优秀的生防菌株^[44]。笔者实验室前期基础研究已完成对Bs916的全基因组精细图谱的测序工作^[36,45],构建转座子标签库,筛选生物膜缺陷表型获取到fliZ插入位点并验证^[34]。本研究以fliZ为切入点,通过生物信息学分析和文献报道,fliZ基因位点位于细菌控制鞭毛运动的fli信号通路,是Ⅱ型正调控因子,与菌体游动能力直接正相关^[46,47]。WANG等^[48]通过缺失表达假结核耶尔森菌YPIII鞭毛系统一级主调控蛋白FlhDC,使其丧失游动性的同时发现其在不同表面上生物膜形成能力显著下降;ZHUO等^[49]研究发现,在柑橘溃疡病菌（Xanthomonas citri subsp. citri）中缺失表达CarB蛋白可导致其在半固体培养基表面游动性显著下降,也导致了突变株在聚苯乙烯孔板中生物膜形成强度的显著下降;SANCHEZ-TORRES等^[50]研究表明,大肠杆菌中环二鸟苷酸（c-di-GMP）含量能够显著负调控游动性与生物膜的形成,其主要由GGDEF蛋白进行负调控,在缺失表达GGDEF蛋白如YeaI、YedQ和YfiN的菌株中均表现出游动性与早期生物膜形成显著增强的现象。本研究构建了fliZ定向敲除突变株,检测了其游动性和生物膜形成能力,结果显示fliZ突变株在上述两表型上均具有显著缺陷,因此笔者认为Bs916中受fliZ调控下的游动性和生物膜形成能力呈正相关。

通过直接分泌抗生素来杀死或抑制病原菌入侵和扩展是生防菌防治植物病害的主要手段^[39,51],本研究检测了fliZ位点突变后对水稻纹枯病菌抑菌能力的变化,发现fliZ突变并不影响其对水稻纹枯病菌的抑制能力。进一步利用HPLC检测了抑菌所需的3种脂肽类抗生素杆菌霉素L、表面活性素和泛革素的相对产量,发现仅有杆菌霉素L的相对产量显著增强,结合抑菌圈直径无显著差异,笔者认为可能是杆菌霉素L在抑菌作用中的贡献较小所致。

定殖能力的强弱是另一个影响芽孢杆菌防治效果的重要因子,传统定殖试验普遍发生在病原菌侵入的寄主植物根部^{[40,41,42,43]},本研究中的水稻纹枯病菌主要入侵水稻茎秆部位,因此借助于绿色荧光蛋白标记技术和激光共聚焦显微镜能够直接检测在接种水稻纹枯病菌的水稻茎秆部位定殖情况,发现fliZ位点突变严重降低了Bs916在水稻茎秆上聚集程度,菌群无序分布,菌体数量低于Bs916,说明其在水稻茎秆上的定殖能力显著下降。芽孢杆菌脂肽抗生素和在寄主体表的定殖能力最终决定对植物病害的防治效果,本研究测定了fliZ位点突变对水稻纹枯病防治效果的影响,发现其防治水稻纹枯病的效果均显著下降,笔者认为是由于其显著下降的生物膜形成能力与在水稻茎秆定殖效果导致的。

4 结论

fliZ位于枯草芽孢杆菌Bs916控制鞭毛游动信号通路中,直接调控菌体的游动和扩展能力,对Bs916生物膜形成具有重要的单一调控作用,在抑菌能力无显著差异前提下直接调控Bs916在水稻茎秆的定殖能力,最终显著调控对水稻纹枯病的防治效果。

参考文献 View Option 原文顺序
文献年度倒序
文中引用次数倒序
被引期刊影响因子

[1] KUMAR K V K, REDDY M S, KLOEPPER J W, LAWRENCE K S, GROTH D E, MILLER M E . Sheath blight disease of rice (Oryza sativa L.)-An overview
Biosciences, Biotechnology Research Asia, 2009,6(2):465-480.
[本文引用: 1]

[2] CHOUDHURY D, ANAND Y R, KUNDU S, NATH R, KOLE R K, SAHA J . Effect of plant extracts against sheath blight of rice caused by Rhizoctonia solani
Journal of Pharmacognosy and Phytochemistry, 2017,6(4):399-404.
[本文引用: 1]

[3] BONANOMI G, CESARANO G, ANTIGNANI V, DI MAIO C, DE FILIPPIS F, SCALA F . Conventional farming impairs Rhizoctonia solani disease suppression by disrupting soil food web
Journal of Phytopathology, 2018,166(9):663-673.
[本文引用: 1]

[4] ZHENG A, LIN R, ZHANG D, QIN P, XU L, AI P, DING L, WANG Y, CHEN Y, LIU Y, SUN Z, FENG H, LIANG X, FU R, TANG C, LI Q, ZHANG J, XIE Z, DENG Q, LI S, WANG S, ZHU J, WANG L, LIU H, LI P . The evolution and pathogenic mechanisms of the rice sheath blight pathogen
Nature Communications, 2013,4:1424.
[本文引用: 1]

[5] LUO C, ZHOU H, ZOU J, WANG X, ZHANG R, XIANG Y, CHEN Z . Bacillomycin L and surfactin contribute synergistically to the phenotypic features of Bacillus subtilis 916 and the biocontrol of rice sheath blight induced by Rhizoctonia solani
Applied Microbiology and Biotechnology, 2015,99(4):1897-1910.
[本文引用: 4]

[6] SHRESTHA B K, KARKI H S, GROTH D E, JUNGKHUN N, HAM J H . Biological control activities of rice-associated Bacillus sp. strains against sheath blight and bacterial panicle blight of rice
PLoS ONE, 2016,11(1):e0146764.
[本文引用: 1]

[7] QI Z, YU J, SHEN L, YU Z, YU M, DU Y, ZHANG R, SONG T, YIN X, ZHOU Y, LI H, WEI Q, LIU Y . Enhanced resistance to rice blast and sheath blight in rice (Oryza sativa L.) by expressing the oxalate decarboxylase protein Bacisubin from Bacillus subtilis
Plant Science, 2017,265:51-60.
[本文引用: 1]

[8] ALETI G, LEHNER S, BACHER M, COMPANT S, NIKOLIC B, PLESKO M, SCHUHMACHER R, SESSITSCH A, BRADER G . Surfactin variants mediate species‐specific biofilm formation and root colonization in Bacillus
Environmental Microbiology, 2016,18(8):2634-2645.
[本文引用: 1]

[9] XU Z, ZHANG H, SUN X, LIU Y, YAN W, XUN W, SHEN Q, ZHANG R . Bacillus velezensis wall teichoic acids are required for biofilm formation and root colonization
Applied and Environmental Microbiology, 2019,85(5):e02116-18.
[本文引用: 1]

[10] GUTTENPLAN S B, BLAIR K M, KEARNS D B . The EpsE flagellar clutch is bifunctional and synergizes with EPS biosynthesis to promote Bacillus subtilis biofilm formation
PLoS Genetics, 2010,6(12):e1001243.
[本文引用: 2]

[11] CZACZYK K, MYSZKA K . Biosynthesis of extracellular polymeric substances (EPS) and its role in microbial biofilm formation
Polish Journal of Environmental Studies, 2007,16(6):799-806.
[本文引用: 1]

[12] MARVASI M, VISSCHER P T, CASILLAS MARTINEZ L . Exopolymeric substances (EPS) from Bacillus subtilis: Polymers and genes encoding their synthesis
FEMS Microbiology Letters, 2010,313(1):1-9.
[本文引用: 1]

[13] DIEHL A, ROSKE Y, BALL L, CHOWDHURY A, HILLER M, MOLIèRE N, KRAMER R, ST?PPLER D, WORTH C L, SCHLEGEL B, LEIDERT M, CREMER N, ERDMANN N, LOPEZ D, STEPHANOWITZ H, KRAUSE E, VAN ROSSUM B J, SCHMIEDER P, HEINEMANN U, TURGAY K, AKBEY ü, OSCHKINAT H . Structural changes of TasA in biofilm formation of Bacillus subtilis
Proceedings of the National Academy of Sciences of the United States of America, 2018,115(13):3237-3242.
[本文引用: 2]

[14] ZHOU H, LUO C, FANG X, XIANG Y, WANG X, ZHANG R, CHEN Z . Loss of GltB inhibits biofilm formation and biocontrol efficiency of
Bacillus subtilis Bs916 by altering the production of γ-polyglutamate and three lipopeptides. PLoS ONE, 2016,11(5):e0156247.
[本文引用: 4]

[15] BISCHOFF D S, WEINREICH M D, ORDAL G W . Nucleotide sequences of Bacillus subtilis flagellar biosynthetic genes fliP and fliQ and identification of a novel flagellar gene, fliZ
Journal of Bacteriology, 1992,174(12):4017-4025.
[本文引用: 1]

[16] XU S J, PENG Z, CUI B, WANG T, SONG Y, ZHANG L, WEI G, WANG Y, SHEN X . FliS modulates FlgM activity by acting as a non-canonical chaperone to control late flagellar gene expression, motility and biofilm formation in Yersinia pseudotuberculosis
Environmental Microbiology, 2014,16(4):1090-1104.
[本文引用: 1]

[17] 杨丙烨, 付丹, 胡方平, 蔡学清 . 西瓜细菌性果斑病菌鞭毛基因fliS的功能分析
中国农业科学, 2017,50(15):2946-2956.
[本文引用: 1]

YANG B Y, FU D, HU F P, CAI X Q . Function analysis of flagellar gene fliS in Acidovorax citrulli.
Scientia Agricultura Sinica, 2017,50(15):2946-2956. (in Chinese)
[本文引用: 1]

[18] BRANDA S S, VIK ?, FRIEDMAN L, KOLTER R . Biofilms: the matrix revisited
TRENDS in Microbiology, 2005,13(1):20-26.
[本文引用: 1]

[19] BRANDA S S, CHU F, KEARNS D B, LOSICK R, KOLTER R . A major protein component of the Bacillus subtilis biofilm matrix
Molecular Microbiology, 2006,59(4):1229-1238.


[20] ROMERO D, AGUILAR C, LOSICK R, KOLTER R . Amyloid fibers provide structural integrity to Bacillus subtilis biofilms
Proceedings of the National Academy of Sciences of the United States of America, 2010,107(5):2230-2234.
[本文引用: 1]

[21] KOBAYASHI K, IWANO M . BslA (YuaB) forms a hydrophobic layer on the surface of Bacillus subtilis biofilms
Molecular Microbiology, 2012,85(1):51-66.
[本文引用: 1]

[22] KEARNS D B, CHU F, BRANDA S S, KOLTER R, LOSICK R . A master regulator for biofilm formation by Bacillus subtilis
Molecular Microbiology, 2005,55(3):739-749.
[本文引用: 1]

[23] DELOUGHERY A, DENGLER V, CHAI Y, LOSICK R . Biofilm formation by Bacillus subtilis requires an endoribonuclease-containing multisubunit complex that controls mRNA levels for the matrix gene repressor SinR
Molecular Microbiology, 2016,99(2):425-437.
[本文引用: 1]

[24] KAMPF J, GERWIG J, KRUSE K, CLEVERLEY R, DORMEYER M, GRüNBERGER A, KOHLHEYER D, COMMICHAU F M, LEWIS R J, STüLKE J, . Selective pressure for biofilm formation in Bacillus subtilis: Differential effect of mutations in the master regulator SinR on bistability
MBio, 2018,9(5):e01464-18.
[本文引用: 1]

[25] HAMON M A, STANLEY N R, BRITTON R A, GROSSMAN A D, LAZAZZERA B A . Identification of AbrB-regulated genes involved in biofilm formation by
Bacillus subtilis. Molecular Microbiology, 2004,52(3):847-860.
[本文引用: 2]

[26] MURRAY E J, STRAUCH M A, STANLEY-WALL N R . σ ^X is involved in controllingBacillus subtilis biofilm architecture through the AbrB homologue Abh.
Journal of Bacteriology, 2009,191(22):6822-6832.
[本文引用: 2]

[27] CHU F, KEARNS D B, BRANDA S S, KOLTER R, LOSICK R . Targets of the master regulator of biofilm formation in Bacillus subtilis
Molecular Microbiology, 2006,59(4):1216-1228.
[本文引用: 1]

[28] STRAUCH M A, BOBAY B G, CAVANAGH J, YAO F, WILSON A, LE BRETON Y . Abh and AbrB control of Bacillus subtilis antimicrobial gene expression
Journal of Bacteriology, 2007,189(21):7720-7732.
[本文引用: 1]

[29] FUJITA M, LOSICK R . Evidence that entry into sporulation in Bacillus subtilis is governed by a gradual increase in the level and activity of the master regulator Spo0A
Genes and Development, 2005,19(18):2236-2244.
[本文引用: 1]

[30] HAMON M A, LAZAZZERA B A . The sporulation transcription factor Spo0A is required for biofilm development in Bacillus subtilis
Molecular Microbiology, 2001,42(5):1199-1209.


[31] DUBNAU E J, CARABETTA V J, TANNER A W, MIRAS M, DIETHMAIER C, DUBNAU D . A protein complex supports the production of Spo0A‐P and plays additional roles for biofilms and the K‐state in Bacillus subtilis
Molecular Microbiology, 2016,101(4):606-624.


[32] BANSE A V, CHASTANET A, RAHN-LEE L, HOBBS E C, LOSICK R . Parallel pathways of repression and antirepression governing the transition to stationary phase inBacillus subtilis
Proceedings of the National Academy of Sciences of the United States of America, 2008,105(40):15547-15552.
[本文引用: 1]

[33] SAIFUDDIN N, WONG C W, YASUMIRA A A . Rapid biosynthesis of silver nanoparticles using culture supernatant of bacteria with microwave irradiation
Journal of Chemistry, 2009,6(1):61-70.
[本文引用: 1]

[34] 周华飞, 罗楚平, 王晓宇, 张荣胜, 陈志谊 . 枯草芽胞杆菌Bs916突变体库的构建和抑制水稻细菌性条斑病菌相关基因的克隆
中国农业科学, 2013,46(11):2232-2239.
[本文引用: 4]

ZHOU H F, LUO C P, WANG X Y, ZHANG R S, CHEN Z Y . Construction of Bacillus subtilis Bs916 mutant libraries by transposon tagging and cloning the genes to the organism’s anti-bacterial activities.
Scientia Agricultura Sinica, 2013,46(11):2232-2239. (in Chinese)
[本文引用: 4]

[35] GRABA M, SAUVAGE S, MOULIN F Y, URREA G, SABATER S, SANCHEZ-PéREZ J M . Interaction between local hydrodynamics and algal community in epilithic biofilm
Water Research, 2013,47(7):2153-2163.
[本文引用: 1]

[36] LUO C, LIU X, ZHOU H, WANG X, CHEN Z . Nonribosomal peptide synthase gene clusters for lipopeptide biosynthesis in Bacillus subtilis 916 and their phenotypic functions
Applied and Environmental Microbiology, 2015,81(1):422-431.
[本文引用: 2]

[37] 周而勋, 曹菊香, 杨媚, 朱西儒 . 我国南方六省(区)水稻纹枯病菌遗传多样性的研究
南京农业大学学报, 2002,25(3):36-40.
[本文引用: 1]

ZHOU E X, CAO J X, YANG M, ZHU X R . Studies on the genetic diversity of Rhizoctonia solani AG-1-IA from six provinces in the southern China.
Journal of Nanjing Agricultural University, 2002,25(3):36-40. (in Chinese)
[本文引用: 1]

[38] 邹成佳, 唐芳, 杨媚, 贺晓霞, 李献军, 周而勋 . 华南3省(区)水稻纹枯病菌的生物学性状与致病力分化研究
中国水稻科学, 2011,25(2):206-212.
[本文引用: 1]

ZOU C J, TANG F, YANG M, HE X X, LI X J, ZHOU E X . Studies on biological characteristics and pathogenicity differentiation of rice sheath blight pathogen from three provinces in South China
Chinese Journal of Rice Science, 2011,25(2):206-212. (in Chinese)
[本文引用: 1]

[39] 向亚萍, 陈志谊, 罗楚平, 周华飞, 刘永锋 . 芽孢杆菌的抑菌活性与其产脂肽类抗生素的相关性
中国农业科学, 2015,48(20):4064-4076.
[本文引用: 2]

XIANG Y P, CHEN Z Y, LUO C P, ZHOU H F, LIU Y F . The antifungal activities of Bacillus spp. and its relationship with lipopeptide antibiotics produced by Bacillus spp
Scientia Agricultura Sinica, 2015,48(20):4064-4076. (in Chinese)
[本文引用: 2]

[40] BIANCIOTTO V, ANDREOTTI S, BALESTRINI R, BONFANTE P, PEROTTO S . Mucoid mutants of the biocontrol strain Pseudomonas fluorescens CHA0 show increased ability in biofilm formation on mycorrhizal and nonmycorrhizal carrot roots
Molecular Plant-Microbe Interactions, 2001,14(2):255-260.
[本文引用: 2]

[41] BAIS H P, FALL R, VIVANCO J M . Biocontrol of Bacillus subtilis against infection of Arabidopsis roots by Pseudomonas syringae is facilitated by biofilm formation and surfactin production
Plant Physiology, 2004,134(1):307-319.
[本文引用: 2]

[42] TIMMUSK S, GRANTCHAROVA N, WAGNER E G H . Paenibacillus polymyxa invades plant roots and forms biofilms
Applied and Environmental Microbiology, 2005,71(11):7292-7300.
[本文引用: 2]

[43] CHEN Y, CAO S, CHAI Y, CLARDY J, KOLTER R, GUO J H, LOSICK R . A Bacillus subtilis sensor kinase involved in triggering biofilm formation on the roots of tomato plants
Molecular Microbiology, 2012,85(3):418-430.
[本文引用: 2]

[44] 陈志谊, 刘永锋, 陆凡 . 水稻纹枯病生防菌Bs-916产业化生产关键技术
植物保护学报, 2004,31(3):230-234.
[本文引用: 1]

CHEN Z Y, LIU Y F, LU F . Study on key technology in the industrialized production of Bacillus subtilis Bs-916, the rice sheath blight control agent.
Acta Phytophylacica Sinica, 2004,31(3):230-234. (in Chinese)
[本文引用: 1]

[45] WANG X, LUO C, CHEN Z . Genome sequence of the plant growth-promoting rhizobacterium Bacillus sp. strain 916
Journal of Bacteriology, 2012,194(19):5467-5468.
[本文引用: 1]

[46] IYODA S, KAMIDOI T, HIROSE K, KUTSUKAKE K, WATANABE H . A flagellar gene fliZ regulates the expression of invasion genes and virulence phenotype in Salmonella enterica serovar Typhimurium
Microbial Pathogenesis, 2001,30(2):81-90.
[本文引用: 1]

[47] KUTSUKAKE K, IKEBE T, YAMAMOTO S . Two novel regulatory genes, fliT and fliZ, in the flagellar regulon of Salmonella
Genes and Genetics Systems, 1999,74(6):287-292.
[本文引用: 1]

[48] WANG Y, DING L S, HU Y B, ZHANG Y, YANG B Y, SHEN S Y . The flhDC gene affects motility and biofilm formation in Yersinia pseudotuberculosis
Science in China Series C: Life Sciences, 2007,50(6):814-821.
[本文引用: 1]

[49] ZHUO T, WEI R, SONG X, GUO J, FAN X, KAMAU G G, ZOU H . Molecular study on the carAB operon reveals that carB gene is required for swimming and biofilm formation in Xanthomonas citri subsp. citri
BMC Microbiology, 2015,15:225.
[本文引用: 1]

[50] SANCHEZ-TORRES V, HU H, WOOD T K . GGDEF proteins YeaI, YedQ, and YfiN reduce early biofilm formation and swimming motility in Escherichia coli
Applied and Microbiology Biotechnology, 2011,90(2):651-658.
[本文引用: 1]

[51] 高学文, 姚仕义, PHAM H, VATER J, 王金生, . 基因工程菌枯草芽孢杆菌GEB3产生的脂肽类抗生素及其生物活性研究
中国农业科学, 2003,36(12):1496-1501.
[本文引用: 1]

GAO X W, YAO S Y, PHAM H, VATER J, WANG J S . Lipopeptide antibiotic produced by the engineered strain Bacillus subtilis GEB3 and detection of its bioactivity.
Scientia Agricultura Sinica, 2003,36(12):1496-1501. (in Chinese)
[本文引用: 1]