贝莱斯芽孢杆菌E69预防稻瘟病等多种真菌病害的潜力

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沙月霞^,¹, 隋书婷², 曾庆超², 沈瑞清¹1 宁夏农林科学院植物保护研究所,银川 750011
2 中国农业大学植物保护学院,北京100193

Biocontrol Potential of Bacillus velezensis Strain E69 Against Rice Blast and Other Fungal Diseases

SHA YueXia^,¹, SUI ShuTing², ZENG QingChao², SHEN RuiQing¹1 Institute of Plant Protection, Ningxia Academy of Agriculture and Forestry Sciences, Yinchuan 750011
2 College of Plant Protection, China Agricultural University, Beijing 100193

通讯作者: 沙月霞,E-mail：yuexiasha@126.com。

责任编辑: 岳梅
收稿日期:2019-01-29接受日期:2019-03-11网络出版日期:2019-06-01

基金资助:

宁夏农林科学院科技先导资金项目.NKYJ-16-26
国家重点研发计划.2017YFD0201606
宁夏回族自治区重点研发计划.2018BBF02018-06
宁夏农林科学院全产业链创新示范项目.QCYL-2018-09

Received:2019-01-29Accepted:2019-03-11Online:2019-06-01
作者简介 About authors
沙月霞,E-mail：yuexiasha@126.com

摘要
目的明确水稻内生菌贝莱斯芽孢杆菌（Bacillus velezensis）菌株E69对多种植物病原真菌的拮抗作用,尤其是对稻瘟病的生物防治效果,减少化学农药的使用。方法贝莱斯芽孢杆菌菌株E69和枯草芽孢杆菌（B. subtilis,稻瘟病生物防治最常用微生物）菌株E66分离于水稻叶片内生细菌,采用对峙培养法测试菌株E69和E66及其发酵液、无菌上清液对稻瘟病菌（Magnaporthe oryzae）的拮抗作用,并测试两株内生芽孢杆菌对立枯丝核菌（Rhizoctonia solani）、镰孢菌（Fusarium spp.）、番茄灰霉病菌（Botrytis cinerea）、草莓炭疽病菌（Colletotrichum gloeospoioides）、烟草黑胫病菌（Phytophthora parasitica var. nicotianae）、链格孢菌（Alternaria alternate）、西瓜枯萎病菌（F. oxysporum）等11种植物病原菌的拮抗作用,温室条件下检测对水稻叶瘟的预防效果,田间试验检测对水稻叶瘟和穗颈瘟的预防效果,常规抑菌测试法研究对稻瘟病菌分生孢子萌发和附着胞形成的抑制作用。采用激光共聚焦显微镜观察绿色荧光蛋白标记后的工程菌株E69在水稻茎部的定殖情况。结果菌株E69和E66对稻瘟病菌菌丝生长具有显著拮抗作用,温室条件下两个菌株对稻瘟病的预防效果分别为83.24%和76.57%,对叶瘟的田间预防效果分别为85.97%和79.76%,对穗颈瘟的田间预防效果分别为69.67%和68.82%,E69对叶瘟的预防效果显著高于75%的三环唑可湿性粉剂,对穗颈瘟的预防效果与三环唑无显著差异。菌株E69和E66对立枯丝核菌、镰孢菌、番茄灰霉病菌、草莓炭疽病菌、烟草黑胫病菌、叶枯病菌、西瓜枯萎病菌等11种植物病原菌有显著拮抗作用,E69的拮抗作用明显高于E66。菌株E69和E66能够强烈抑制稻瘟病菌分生孢子萌发和附着胞的形成,E69发酵液的抑菌效果分别为95.28%和94.16%,无菌上清液的抑菌效果分别为85.36%和84.31%;E66发酵液对分生孢子萌发和附着胞形成的抑菌效果分别为89.15%和87.38%,无菌上清液的抑菌效果分别为79.65%和72.45%。绿色荧光蛋白GFP78标记后的工程菌株E69在水稻茎部具有较好的定殖能力,可以稳定定殖在水稻茎部表皮、薄壁组织和维管束。结论贝莱斯芽孢杆菌菌株E69是一种潜在的、预防效果明显的生防菌株,具有预防稻瘟病兼防纹枯病等多种真菌病害的应用潜力。
关键词： 贝莱斯芽孢杆菌;稻瘟病;植物病原真菌;生物防治;定殖能力

Abstract

【Objective】 The objective of this study is to clarify the antagonistic effect of Bacillus velezensis strain E69 isolated from rice endophytic bacteria against various fungal plant pathogens in vitro, especially the control efficacy of B. velezensis E69 on rice blast, and to reduce the use of chemical pesticides.【Method】B. velezensis E69 and B. subtilis (most commonly used microorganisms in biological control of rice blast) E66 strains were isolated from endophytic bacteria of rice leaf tissue. The antagonistic effect of strains E69 and E66 and their fermented liquid, sterile supernatant against Magnaporthe oryzae was tested by confrontation culture method. Also, the antagonistic effect of strains E69 and E66 against other 11 pathogenic fungi, such as Rhizoctonia solani, Fusarium spp., Botrytis cinerea, Colletotrichum gloeospoioides, Phytophthora parasitica var. nicotianae, Alternaria alternate, F. oxysporum, et al., was determined. The preventive efficacy of E69 and E66 was tested for the control of rice leaf blast under greenhouse condition. The field experiments were conducted to evaluate the preventive efficacy of E69 and E66 against rice leaf blast and neck blast. The inhibitory effect of E69 and E66 against conidial germination and appressorial formation of M. oryzae was assessed in laboratory. The colonization of GFP-marked strain E69 in rice stem was observed by laser scanning confocal microscope.【Result】 Strains E69 and E66 significantly suppressed the mycelia growth rate of M. oryzae P131, the preventive efficacy against rice blast under greenhouse condition was 83.24% and 76.57%, respectively. The preventive efficacy of E69, E66 against rice leaf blast and neck blast in field was 85.97%, 79.76% and 69.67%, 68.82%, respectively. The preventive effect of E69 on leaf blast was significantly higher than that of 75% tricyclozole wettable powder, but there was no significant difference between the preventive efficacy of strain E69 and 75% tricyclazole powder against rice neck blast. E69 and E66 had significant antagonistic effects against 11 plant pathogens, such as R. solani, Fusarium spp., B. cinerea, C. gloeospoioides, P. parasitica var. nicotianae, A. alternate, F. oxysporum, et al. The antagonistic effect of E69 was higher than that of E66. E69 and E66 could strongly inhibit the conidial germination and appressorial formation of M. oryzae P131, the inhibitory effect of E69 fermented liquid was 95.28% and 94.16%, respectively, and the inhibitory effect of E69 sterile supernatant was 85.36% and 84.31%, respectively. The inhibitory effect of E66 fermented liquid was 89.15% and 87.38%, respectively, and the inhibitory effect of E66 sterile supernatant were 79.65% and 72.45%, respectively. The GFP-marked strain E69 showed good colonization ability in rice stem, and could be stably colonized in rice stem epidermis, parenchyma and vascular bundles. 【Conclusion】B. velezensis strain E69 is a potential biocontrol strain with obvious preventive effects, which has the application potential of preventing rice blast, sheath blight and other fungal diseases.

Keywords：Bacillus velezensis;rice blast;plant pathogenic fungi;biological control;colonization ability

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本文引用格式
沙月霞, 隋书婷, 曾庆超, 沈瑞清. 贝莱斯芽孢杆菌E69预防稻瘟病等多种真菌病害的潜力[J]. 中国农业科学, 2019, 52(11): 1908-1917 doi:10.3864/j.issn.0578-1752.2019.11.006
SHA YueXia, SUI ShuTing, ZENG QingChao, SHEN RuiQing. Biocontrol Potential of Bacillus velezensis Strain E69 Against Rice Blast and Other Fungal Diseases[J]. Scientia Acricultura Sinica, 2019, 52(11): 1908-1917 doi:10.3864/j.issn.0578-1752.2019.11.006

0 引言

【研究意义】水稻是世界范围内重要的粮食作物,稻瘟病（rice blast）是危害水稻生产最严重的病害,其病原菌的无性态为无性菌类丝孢目梨孢属灰梨孢菌Pyricularia oryzae,有性态为子囊菌门巨座壳目巨座壳属Magnaporthe oryzae^[1]。稻瘟病发生范围广泛,平均每年造成20%—30%的产量损失,流行年份损失达到50%以上,严重的甚至绝产^[2]。水稻抗病品种选育、农艺措施和化学农药防治是目前生产上最常用的措施,由于稻瘟病菌生理小种易变异,抗病品种选育一般时间较长;化学农药的使用容易使病原菌产生抗药性且对生态环境和人类健康有一定影响。因此,寻找一种安全、环境友好并具有良好防治效果的新型生防菌对水稻产业可持续发展具有重要意义。【前人研究进展】芽孢杆菌产生抗逆性极强的芽孢,对生态环境无污染,可以与环境中的其他微生物互作,具有广谱的抑菌活性,诱导植株产生抗病性,是稻瘟病防治研究的热点之一^[3]。目前在稻瘟病防治中应用较多的芽孢杆菌包括枯草芽孢杆菌（Bacillus subtilis）^[4]、解淀粉芽孢杆菌（B. amyloliquefaciens）^[5]、短小芽孢杆菌（B. pumilus）^[6]、地衣芽孢杆菌（B. licheniformis）、多粘类芽孢杆菌（Panebacillus polymyxa）和坚强芽孢杆菌（B. firmus）等。但是已报道的芽孢杆菌大多来源于根际促生菌（plant growth promoting rhizobacteria）^[7]、土壤^[8]和植株表面^[9],较少来源于植物内生菌。植物内生菌是植物微生态系统的重要组成部分^[10,11],具有非常重要的生态功能^[12,13],包括具有较强且广谱的抑菌防虫作用^[14,15]、促生增产作用^[16,17]、促进植株体内氮等营养元素的转化与吸收^[18,19]、增强寄主抗逆性^[20,21]、降解重金属污染和农药残留^[22]等,可以用于研制生物杀菌剂^[23]和微生物肥料^[24]。关于植物内生菌防治稻瘟病的研究国内外虽有不少报道,但是植物内生芽孢杆菌防治稻瘟病的研究尚处于探索阶段。JHA等^[25]研究表明,分离于水稻根系内生菌的类产碱假单胞菌（Pseudomonas pseudoalcaligenes）GJ17对稻瘟病菌的生防效果明显高于根际促生菌短小芽孢杆菌;ZHU等^[26]研究表明,内生真菌柿假尾胞菌（Pseudocercospora kaki）和菌核青霉（Penicillium sclerotiorum）对稻瘟病菌菌丝生长的抑制率分别达78.02%和62.81%。生防微生物在植株上的定殖、扩散和生存竞争能力是考核生防作用的重要指标之一^[27,28]。【本研究切入点】植物内生菌贝莱斯芽孢杆菌（B. velezensis）菌株E69为笔者实验室前期从宁夏回族自治区水稻叶片中分离得到,其对稻瘟病菌、立枯丝核菌（Rhizoctonia solani）等多种病原真菌的拮抗作用和生防应用潜力有待明确。【拟解决的关键问题】探明贝莱斯芽孢杆菌菌株E69对稻瘟病的预防效果和对立枯丝核菌等11种病原真菌的拮抗作用,明确其对稻瘟病菌孢子萌发和附着胞形成的抑制作用以及在水稻茎部的定殖情况,为应用E69菌株预防稻瘟病、纹枯病、灰霉病、叶枯病等真菌病害提供理论依据。

1 材料与方法

1.1 试验田概况

田间试验于2018年在宁夏回族自治区平罗县姚伏镇（106.52°E,38.91°N）水稻种植主栽地区进行。试验地常年种植水稻,稻瘟病每年普遍发生,行距为30 cm,正常肥水管理,试验期间不喷施其他杀菌剂。土壤类型为草甸土,碳酸钙含量5—78 g·kg^-1,全磷含量：0.32—0.49 g·kg^-1,全钾含量：16.7—29.5 g·kg^-1,全氮含量：0.14—0.94 g·kg^-1,pH 7.2—8.8,有机质含量1.2—13.1 g·kg^-1。

1.2 生防菌、病原菌及其培养

稻瘟病菌P131由中国农业大学植物保护学院提供。尖镰孢（Fusarium oxysporum）N16-2-1、茄镰孢（F. solani）N18-1-2、串珠镰孢（F. moniliforme）N19-2-2、立枯丝核菌RS8由笔者实验室分离;西瓜枯萎病菌（F. oxysporum f. sp. niveum）M8、番茄灰霉病菌（Botrytis cinerea）ZDP4、草莓炭疽病菌（Colletotrichum gloeospoioides）ZDP21、苹果叶枯病菌链格孢菌（Alternaria alternate）BJ-A5、海棠叶枯病菌链格孢菌BJ-H9、烟草黑胫病菌（Phytophthora parasitica var. nicotianae）T15、草莓叶枯病菌链格孢菌BJ-ST24由北京市农林科学院植物保护环境保护研究所分离。田间试验水稻品种宁粳47,温室试验水稻品种G19,均为宁夏水稻种植地区主栽品种。

水稻内生芽孢杆菌菌种：贝莱斯芽孢杆菌E69和枯草芽孢杆菌E66是从宁夏回族自治区水稻叶片中分离得到的内生菌,GFP标记菌株为采用绿色荧光蛋白GFP78标记后的E69。

2个水稻内生芽孢杆菌在牛肉膏蛋白胨固体培养基平板上活化,在37℃黑暗条件下培养。稻瘟病菌P131的活化和培养在番茄燕麦培养基上进行。11种其他植物病原真菌菌株在PDA平板上活化和培养。

1.3 对峙培养

在番茄燕麦平板培养基中央放置稻瘟病菌菌饼（1 cm）,待稻瘟病菌菌落生长2 d后,分别在菌落边缘相距2 cm处接种芽孢杆菌菌饼（5 mm）,有一侧为空白对照,置于培养箱28℃黑暗培养,4次重复。4 d后测量病原菌菌落直径。

在PDA培养基平板上中央放置上述11种植物病原菌的菌饼（1 cm）,分别在菌落边缘两侧相距2 cm处接种芽孢杆菌菌株（划线2 mm）,置于培养箱28℃黑暗培养,5 d后测量病原菌菌落直径和抑菌带。

1.4 水稻内生芽孢杆菌对稻瘟病的防治效果

温室试验设计5个处理：处理Ⅰ-CK为清水对照;Ⅱ-菌株E66发酵液;Ⅲ-菌株E69发酵液; Ⅳ-75%三环唑可湿性粉剂（陶氏益农农业科技（中国）有限公司）;Ⅴ-绿地康1号（中农绿康（北京）生物技术有限公司）。每处理4个重复。水稻种植28 d后,CK喷施无菌水,处理Ⅱ、Ⅲ喷施内生芽孢杆菌发酵液,喷雾芽孢浓度为1×10⁸ CFU/mL,处理Ⅳ喷施20%的三环唑水溶液,处理Ⅴ喷施绿地康1号的100倍稀释液,每个水桶种植5株水稻,喷施20 mL。24 h后所有处理均喷施20 mL稻瘟病菌孢子悬浮液（1×10⁶CFU/mL）。7 d后调查叶瘟发生情况。

田间试验地点在平罗县姚伏镇,试验设计5个处理：CK为清水对照;菌株E66和E69,喷雾芽孢浓度为1×10⁸CFU/mL;75%三环唑可湿性粉剂;绿地康1号。分别在水稻插秧后30、45、60、75和90 d叶面喷施菌剂发酵液、75%三环唑可湿性粉剂和绿地康1号。75%三环唑可湿性粉剂的用药量为1.05—1.20 kg·hm^-2,药液量为600—750 kg·hm^-2。绿地康1号的用药量为7.50 L·hm^-2（有效活菌数≥1×10⁹/mL）,药液量为2 400 kg·hm^-2。稻瘟病在试验田每年均有发生,插秧后45 d调查试验田叶瘟的发生情况,插秧后100 d调查穗颈瘟的发生状况,调查分级标准采用中华人民共和国国家标准（GB/T15970—1995）。试验小区采取随机排列,小区平均面积50 m²,每处理4个重复小区。

1.5 水稻内生芽孢杆菌对稻瘟病菌孢子萌发的抑制作用

枯草芽孢杆菌E66和贝莱斯芽孢杆菌E69的发酵液（芽孢浓度为1×10⁸CFU/mL）、无菌上清液（发酵液离心后的上清液经0.22 μm抽滤）分别与稻瘟病菌孢子悬浮液（1×10⁶ CFU/mL）以1﹕1的比例混匀,混合液在无菌12孔板中28℃对峙培养24 h,在激光共聚焦显微镜下观察稻瘟病菌P131分生孢子萌发和附着胞形成情况,每次观察200个分生孢子。

1.6 贝莱斯芽孢杆菌E69在水稻植株上的定殖

水稻种植20 d时,用已成功标记绿色荧光蛋白GFP的贝莱斯芽孢杆菌E69发酵液（芽孢浓度为1×10⁸ CFU/mL）喷施植株,7 d后用LEICA 3050S冷冻切片机做水稻茎部横切面和纵切面的冷冻切片,厚度约20 μm,用LEICATCS SP8激光共聚焦显微镜观察供试菌株在水稻植株的定殖情况。激发光是绿光,波长为488 nm,采用Photoshop11.0图片编辑软件编辑照片。

1.7 数据处理与分析

所有数据在Excel 2007中计算平均值±标准误,采用DPS15.0统计软件进行分析,方差分析选用单因素分析中的最长距离法计算（P<0.05）。

计算公式：病情指数=[∑各级病叶（穗）数×相对级数值]/[调查总叶（穗）数×9]×100;预防效果（%）=[（CK 病情指数-处理病情指数）/CK病情指数]×100。

2 结果

2.1 水稻内生芽孢杆菌对稻瘟病菌的拮抗作用

水稻内生芽孢杆菌菌株E69和E66均能够显著抑制稻瘟病菌P131的生长,拮抗作用明显,相对菌丝抑制率分别达到90.36%和92.56%,其发酵液和无菌上清液的抑菌作用也较为明显（图1）。

图1

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图1水稻内生芽孢杆菌对稻瘟病菌P131菌丝生长的抑制效果

A：稻瘟病菌P131生长9 d的菌落形态The colony morphology of M. oryzae P131 growing for 9 d;B：拮抗菌E69与稻瘟病菌P131对峙培养The colony of M. oryzae P131 inhibited by strain E69;C：拮抗菌E66与稻瘟病菌P131对峙培养The colony of M. oryzae P131 inhibited by strain E66;D：稻瘟病菌P131生长6 d的菌落形态The colony morphology of M. oryzae P131 growing for 6 d;E：拮抗菌E69发酵液与稻瘟病菌P131对峙培养The colony of M. oryzae P131 inhibited by fermented liquid of strain E69;F：拮抗菌E69无菌上清液与稻瘟病菌P131对峙培养The colony of M. oryzae P131 inhibited by sterile supernatant of strain E69;G：拮抗菌E66发酵液与稻瘟病菌P131对峙培养The colony of M. oryzae P131 inhibited by fermented liquid of strain E66;H：拮抗菌E66无菌上清液与稻瘟病菌P131对峙培养The colony of M. oryzae P131 inhibited by sterile supernatant of strain E66
Fig. 1The mycelia growth rate of M. oryzae P131 inhibited by endophytice bacteria of rice

2.2 水稻内生芽孢杆菌对稻瘟病的预防效果

2株水稻内生芽孢杆菌在温室条件下对稻瘟病有显著预防效果（表1）,贝莱斯芽孢杆菌E69对稻瘟病的预防效果为83.24%,与对照生物农药绿地康的预防效果（84.51%）无显著差异（P>0.05）,显著高于化学农药75%三环唑可湿性粉剂的预防效果（77.91%）（P<0.05）。枯草芽孢杆菌E66的预防效果与三环唑无显著差异（P>0.05）,显著低于菌株E69的预防效果（P<0.05）。

Table 1
表1
表1水稻内生芽孢杆菌在温室条件下对稻瘟病的预防效果
Table 1The preventive efficacy of rice endophytic bacteria against rice blast under greenhouse condition

处理Treatment	病情指数Disease index	预防效果Preventive efficacy (%)
E69	3.71±1.11b	83.24±1.41a
E66	5.18±1.05b	76.57±1.15b
75%三环唑Tricyclazole	4.88±1.14b	77.91±1.43b
绿地康Lvdikang	3.42±0.52b	84.51±0.69a
清水对照Water control	22.09±2.43a	—

Different lowercases after the data indicate significantly different (P<0.05). The same as below
数据后不同小写字母表示差异显著（P<0.05）。下同

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2株水稻内生芽孢杆菌对叶瘟的田间预防效果显著（表2）。贝莱斯芽孢杆菌E69预防水稻叶瘟的效果达到85.97%,与绿地康的预防效果无显著性差异（P>0.05）,与三环唑的预防效果差异显著（P<0.05）。菌株E66对叶瘟的田间预防效果与三环唑无显著性差异（P>0.05）。各处理对穗颈瘟的效果无显著性差异（P>0.05）。对穗颈瘟的田间预防效果较低,分析原因主要是试验田在施肥管理上出现失误,生产过程中误施过量尿素,导致穗颈瘟大发生,影响了E69的田间应用效果。

Table 2
表2
表2水稻内生芽孢杆菌预防稻瘟病的田间效果
Table 2The preventive efficacy of rice endophytic bacteria against rice blast in the field

处理 Treatment	叶瘟Rice leaf blast		穗颈瘟Rice neck blast
处理 Treatment	病情指数 Disease index	预防效果 Preventive efficacy (%)	病情指数 Disease index	预防效果 Preventive efficacy (%)
E69	4.66±0.53b	85.97±0.96a	16.56±2.62b	69.67±2.41a
E66	8.79±2.53b	79.76±2.96b	16.48±4.66b	68.82±4.41a
绿地康Lvdikang	4.04±1.29b	88.74±2.65a	16.03±1.77b	69.67±1.54a
75%三环唑Tricyclazole	10.06±2.19b	77.01±2.01b	15.76±1.15b	70.18±1.08a
清水对照Water control	36.15±2.25a	—	52.86±3.06a	—

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2.3 水稻内生芽孢杆菌对立枯丝核菌等病原菌的拮抗作用

2株水稻内生芽孢杆菌对多种植物病原菌具有显著抑菌作用（表3）。贝莱斯芽孢杆菌E69对尖镰孢、茄镰孢和串珠镰孢的抑菌效果>80%,对立枯丝核菌、番茄灰霉病菌、苹果叶枯病菌和海棠叶枯病菌的抑菌效果在71%—80%,对草莓炭疽病菌、西瓜枯萎病菌和烟草黑胫病的抑菌效果在61%—70%,对草莓叶枯病菌的抑菌效果<60%。枯草芽孢杆菌E66对立枯丝核菌的抑菌效果在71%—80%,对尖镰孢、苹果叶枯病菌、海棠叶枯病菌和西瓜枯萎病菌的抑菌效果在61%—70%,对其他病原菌的抑菌效果<60%。枯草芽孢杆菌E66对11种植物病原菌具有拮抗作用,但是抑菌效果明显小于菌株E69。

Table 3
表3
表3菌株E69和E66对多种植物病原菌的拮抗作用
Table 3The antagonistic effect of strains E69 and E66 against various fungal plant pathogens in vitro

病原菌 Target fungal pathogen	抑菌效果 Antagonistic effect
病原菌 Target fungal pathogen	E66	E69
Fusarium oxysporum N16-2-1	++	++++
Fusarium solani N18-1-2	+	++++
Fusarium moniliforme N19-2-2	+	++++
Rhizoctonia solani RS8	+++	+++
Botrytis cinerea ZDP4	+	+++
Colletotrichum gloeospoioides ZDP21	+	++
Alternaria alternate BJ-A5	++	+++
Alternaria alternate BJ-ST24	+	+
Alternaria alternate BJ-H9	++	+++
Fusarium oxysporum f. sp. niveum M8	++	++
Phytophthora parasitica var. nicotianae T15	+	++

+, ++, +++ and ++++ represent relative inhibition rates against mycelia growth of each fungal colony on the PDA medium to the extent of 50%- 60%, 61%-70%, 71%-80% and >80%, respectively
+、++、+++和++++代表相对菌丝抑制率分别为50%—60%、61%—70%、71%—80%和>80%

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2.4 水稻内生芽孢杆菌对稻瘟病菌孢子萌发和附着胞形成的抑制作用

2株水稻内生芽孢杆菌对稻瘟病菌孢子萌发和附着胞形成具有显著的抑制作用（表4）。贝莱斯芽孢杆菌E69发酵液对稻瘟病菌分生孢子萌发和附着胞形成的抑制作用分别是95.28%和94.16%,无菌上清液的抑制作用分别是85.36%和84.31%。枯草芽孢杆菌E66发酵液和无菌上清液对稻瘟病菌分生孢子萌发和附着胞形成也具有明显抑制作用,但是抑菌效果较菌株E69低。

Table 4
表4
表4水稻内生芽孢杆菌对稻瘟病菌孢子萌发和附着胞形成的抑制作用
Table 4Inhibitory activity of rice endophytic bacteria against conidial germination and appressorial formation of M. oryzae

处理 Treatment	分生孢子Conidium		附着胞Appressorium
处理 Treatment	萌发率 Germination rate (%)	抑制率 Inhibition rate (%)	形成率 Formation rate (%)	抑制率 Inhibition rate (%)
E69发酵液E69 fermented liquid	4.67±0.82d	95.28±0.83a	5.02±1.14d	94.16±0.15a
E69无菌上清液E69 sterile supernatant	14.49±0.19c	85.36±0.19bc	13.49±0.48bc	84.31±10.55b
E66发酵液E66 fermented liquid	10.74±1.73c	89.15±1.23ab	10.85±0.36c	87.38±0.41b
E66无菌上清液E66 sterile supernatant	20.15±3.44b	79.65±3.47c	23.69±1.59b	72.45±1.86c
清水对照Water control	99.00±0.76a	—	86.00±2.67a	—

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2.5 贝莱斯芽孢杆菌E69在水稻茎部的定殖

采用CLSM观察贝莱斯芽孢杆菌E69在水稻茎部的定殖（图2）。喷施后第7天,在水稻茎部检测到菌株E69的GFP绿色荧光。在纵切面的表皮、基本组织和维管束中观察到标记菌株,尤其是表皮的绿色荧光较强,向内逐步减弱。在横切面的表皮、薄壁组织和中央维管束系统中也观察到的绿色荧光,强弱程度与纵切面一致。说明E69从表皮进入茎部组织,向薄壁组织和中央维管束系统扩展迁移。在无菌水喷雾处理的水稻茎部组织中未发现有荧光标记的E69。

图2

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图2GFP标记后的菌株在水稻茎部定殖的共聚焦图像

Fig. 2Images from a confocal microscope of rice stems inoculated with GFP-marked strains

3 讨论

芽孢杆菌对植物病原菌的生防机制主要包括竞争作用、拮抗作用和诱导抗病性,有的菌株以一种生防机制为主,有的菌株则以多种生防机制协同作用相互增强了对病原菌的拮抗作用^[29]。芽孢杆菌具有防治植物病害的优良特性,在世界范围内被广泛用作商品化的生防制剂。一般来源于植物内部组织的芽孢杆菌抗菌谱较广,抑菌效果明显,受环境因素影响较小,是广大植物病理工作者研究的热点之一。但是目前植物内生芽孢杆菌的商品化研发还处于探索阶段,获得对多种植物病原菌具有拮抗作用的内生芽孢杆菌并用做生防制剂,一直是植物病害生物防治研究的重要内容。

目前,在水稻生产上用于稻瘟病生物防治的芽孢杆菌主要包括枯草芽孢杆菌、解淀粉芽孢杆菌、短小芽孢杆菌、地衣芽孢杆菌等,已有研究表明贝莱斯芽孢杆菌对多种植物病原菌有较强的拮抗作用^[30,31],但国内还未有****研究过其对稻瘟病菌的拮抗作用,本研究证实了贝莱斯芽孢杆菌对稻瘟病具有较明显的预防效果,不仅为稻瘟病杀菌剂的研发提供了新型微生物资源,而且拓宽了贝莱斯芽孢杆菌的应用领域。生防芽孢杆菌大多来源于植株根际、非根际土壤或植株体表,而采用植物内生菌预防稻瘟病的研究报道较少。贝莱斯芽孢杆菌E69和枯草芽孢杆菌E66菌株均来源于水稻叶片内生菌,研究结果进一步印证了植物内生菌具有广泛的生物活性,具备应用于防治稻瘟病等多种真菌病害的生防潜力。

水稻内生芽孢杆菌对稻瘟病菌分生孢子萌发和附着胞形成具有显著抑菌作用,贝莱斯芽孢杆菌E69发酵液几乎完全抑制孢子萌发和附着胞的形成;无菌上清液的抑菌作用也较明显,对孢子萌发和附着胞形成的抑菌率达到84%以上。此结果进一步证实拮抗作用是E69菌株防治稻瘟病的机制之一。芽孢杆菌对病原菌的拮抗作用主要包括抑菌作用和溶菌作用^[32,33],之所以会产生不同的拮抗机制源于菌株产生的抗菌活性物质,包括酶类、细菌素、脂肽类以及挥发性物质等^[34]。之前的研究表明,贝莱斯芽孢杆菌E69产生蛋白酶、淀粉酶、纤维素酶和葡聚糖酶,挥发性物质对稻瘟病菌菌丝生长的抑制效果达到95%以上,但具体哪类抗菌活性物质抑制了孢子萌发和附着胞形成有待于深入研究。

在稻瘟病的田间防治中,一般环境中微生物会与生防菌竞争营养、空气和生态位点,可能会影响生防菌在植株体内的定殖,进而导致防治效果下降^[35,36]。而植物内生生防菌与寄主体内的微生物相容性好,比土壤和其他来源的生防菌在植物体内更容易定殖、扩展和迁移。大量的研究证实,生防菌能否有效在植株体内定殖是生防效果能否稳定、持效的重要因素^[37,38]。本研究检测了绿色荧光蛋白（GFP）标记后的贝莱斯芽孢杆菌E69在水稻茎部的定殖状况,在水稻表皮组织、薄壁组织和维管束中具有较好的定殖能力。GFP是广泛应用的标记分子,不会改变细胞的结构和活性^[39],对细胞无毒,常被研究人员作为标记基因用来研究微生物在植株体内的定殖规律^[40]。笔者还研究了GFP标记后的菌株与原始菌株之间在生长曲线、抑菌作用之间的差异,证实GFP标记的工程菌株与原始菌株在抑菌活性和生长动力学方面没有明显差异（另文发表）,因此可以用GFP作为标记基因研究贝莱斯芽孢杆菌在水稻植株上的定殖情况。

筛选并利用具有多种拮抗机制的芽孢杆菌作为生防菌剂是植物病害生物防治研究的重要内容。NG等^[41]研究表明,解淀粉芽孢杆菌UPMS3和多种微生物混配的微生物菌肥可以显著提高生防菌的活性;KANJANAMANEESATHIAN等^[42]将巨大芽孢杆菌（B. megaterium）浸种和叶面喷雾,结合施用氮肥,可以有效预防穗颈瘟;KYUNG-SEOK等^[43]发现萎缩芽孢杆菌（B. vallismortis）EXTN-1诱导作物PR基因的表达,进而诱导产生抗病性,防治稻瘟病效果达52.11%。本研究结果证实了贝莱斯芽孢杆菌E69具有广谱抗菌活性,温室条件下对稻瘟病的预防效果明显,为应用于稻瘟病的防治提供了依据。田间大区试验进一步证实,E69预防水稻叶瘟的效果较好,与绿地康的预防效果相当,对穗颈瘟也有一定的效果,显示出E69良好的生防应用价值。另外,对11种植物病原真菌的菌丝抑制试验表明,该菌株作为生防制剂可能同时具有防治纹枯病、番茄灰霉病等多种真菌病害的潜力。

4 结论

贝莱斯芽孢杆菌菌株E69对稻瘟病具有明显的生防效果,并且具有广谱的抑菌活性,能够稳定定殖在水稻植株体内,具有在稻瘟病的田间防治中推广应用的潜力。E69发酵液和无菌上清液均可显著抑制稻瘟病菌的分生孢子萌发和附着胞形成。因此,贝莱斯芽孢杆菌E69是一种潜在的、有效的生防菌株,具有防治稻瘟病、纹枯病、叶枯病、炭疽病、灰霉病、土传病害和烟草黑胫病等多种真菌病害的潜力。

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沙月霞, 王琦, 李燕 . 稻瘟病生防芽胞杆菌的筛选及防治效果
中国生物防治学报, 2016,32(4):474-484.

[本文引用: 1]

SHA Y

, WANG

, LI

. Screening and prevention of Bacillus biocontrol against rice blast
Chinese Journal of Biological Control, 2016,32(4):474-484. (in Chinese)

[本文引用: 1]

[10]

TAGHAVI

, GARAFOLA

, MONCHY

, NEWMAN

, HOFFMAN

, WEYENS

, BARAC

, VANGRONSVELD

, VAN DER

LELIE D

. Genome survey and characterization of endophytic bacteria exhibiting a beneficial effect on growth and development of poplar trees
Applied and Environmental Microbiology, 2009,75(3):748-757.

DOI:10.1128/AEM.02239-08 URL [本文引用: 1]

[11]

PRASANNA

, NAIN

, PANDEY A

, SAXENA A

. Microbial diversity and multidimensional interactions in the rice ecosystem
Archives of Agronomy and Soil Science, 2012,58(7):723-744.

DOI:10.1080/03650340.2010.537325 URL [本文引用: 1]

[12]

TANG

, PURI

, PADDA K

, CHANWAY C

. Biological nitrogen fixation and plant growth promotion of lodgepole pine by an endophytic diazotrophPaenibacillus polymyxa and its GFP-tagged derivative
Botany, 2017,95(6):611-619.

DOI:10.1139/cjb-2016-0300 URL [本文引用: 1]

[13]

JI S

, GURURANI M

, CHUN S

. Isolation and characterization of plant growth promoting endophytic diazotrophic bacteria from Korean rice cultivars
Microbiological Research, 2014,169(1):83-98.

DOI:10.1016/j.micres.2013.06.003 URL [本文引用: 1]

[14]

OWNLEY B

, GWINN K

, VEGA F

. Endophytic fungal entomopathogens with activity against plant pathogens: ecology and evolution
BioControl, 2010,55(1):113-128.

DOI:10.1007/s10526-009-9241-x URL [本文引用: 1]

[15]

LIU

, BAI F

, LI

, WANG W

, CHENG

. Identification of endophytic bacterial strain RSE1 from seeds of super hybrid rice Shenliangyou 5814 (Oryza sativa L.) and evaluation of its antagonistic activity
Plant Growth Regulation, 2017,82(3):403-408.

DOI:10.1007/s10725-017-0265-4 URL [本文引用: 1]

[16]

SHYLLA

, SHIVAPRAKASH M

, SHASHIDHAR H

, VISHWAKARMA

, SUDRADHAR

. Production of phytohormones by endophytic bacteria isolated from aerobic rice
Journal of Pure and Applied Microbiology, 2016,10(3):2127-2133.

[本文引用: 1]

[17]

SHAHZAD

, WAQAS

, KHAN A

, AL-HOSNI

, KANG S

, SEO C

, LEE I

. Indoleacetic acid production and plant growth promoting potential of bacterial endophytes isolated from rice (Oryza sativa L.) seeds
Acta Biologica Hungarica, 2017,68(2):175-186.

DOI:10.1556/018.68.2017.2.5 URL [本文引用: 1]

[18]

杨波, 陈晏, 李霞, 任承钢, 戴传超 . 植物内生菌促进宿主氮吸收与代谢研究进展
生态学报, 2013,33(9):2656-2664.

DOI:10.5846/stxb201202050147 Magsci [本文引用: 1]

内生菌与植物共生能够提高宿主的氮吸收与氮代谢水平,这可能是由于内生菌在植物体内引发的多种效应的综合结果.植物内生菌能够通过促进植物根系发育和固氮作用为宿主植物提供更多的无机氮素;能够通过分泌多种胞外酶系如漆酶、蛋白水解酶等使宿主植物更好地利用有机氮素;能够提高宿主氮代谢关键酶如硝酸还原酶(NR)、谷氨酰胺合成酶(GS)等酶的活性;能够提高宿主植物激素水平和维生素含量从而促进宿主氮代谢;能够通过影响宿主植物氮代谢促进宿主植物分蘖、提高宿主植物叶绿素含量和光合速率等等.综述了国内外关于植物内生菌促进宿主氮代谢的相关报道,归纳了植物内生菌影响宿主氮素吸收与代谢的可能机制,并展望了关于植物内生菌促进宿主氮代谢机制方面的研究方向.

YANG

, CHEN

, LI

, REN C

, DAI C

. Research progress on endophyte-promoted plant nitrogen assimilation and metabolism
Acta Ecologica Sinica, 2013,33(9):2656-2664. (in Chinese)

DOI:10.5846/stxb201202050147 Magsci [本文引用: 1]

RANGJAROEN

, RERKASEM

, TEAUMROONG

, SUNGTHONG

, LUMYONG

. Comparative study of endophytic and endophytic diazotrophic bacterial communities across rice landraces grown in the highlands of northern Thailand
Archives of Microbiology, 2014,196(1):35-49.

DOI:10.1007/s00203-013-0940-4 URL [本文引用: 1]

[20]

PHAM V T

, REDIERS

, GHEQUIRE M G

, NGUYEN H

, DE MOT

, VANDERLEYDEN

, SPAEPEN

. The plant growth-promoting effect of the nitrogen-fixing endophytePseudomonas stutzeri A15
Archives of Microbiology, 2017,199(3):513-517.

DOI:10.1007/s00203-016-1332-3 URL [本文引用: 1]

[21]

SHAHZAD

, KHAN A

, BILAL

, WAQAS

, KANG S

, LEE I

. Inoculation of abscisic acid-producing endophytic bacteria enhances salinity stress tolerance in Oryza sativa
Environmental and Experimental Botany, 2017,136:68-77.

DOI:10.1016/j.envexpbot.2017.01.010 URL [本文引用: 1]

[22]

KLAYRAUNG

, NIAMSUP

, POONNOY

, TOPOONYANONT

. Diversity and control of bacterial contamination of plants propagated in temporary immersion bioreactor system
Acta Horticulturae, 2017,1155:439-446.

[本文引用: 1]

[23]

, LI

, ZENG X

, YANG X

, YANG Y

, YUAN S

, HU X

, ZENG J

, WANG Z

, LIU

, LIU Y

, LIAO H

, TONG C

, LIU X

, ZHU Y

. Isolation and evaluation of endophytic Streptomyces endus OsiSh-2 with potential application for biocontrol of rice blast disease.
Journal of the Science of Food and Agriculture, 2017,97(4):1149-1157.

DOI:10.1002/jsfa.7841 URLPMID:27293085 [本文引用: 1]

Abstract Background: Biocontrol is a promising strategy in the control of rice blast disease. In this study, we isolated and characterized a novel antagonist to the pathogen Magnaporthe oryzae from rice endophytic actinomycetes. Results: Out of 482 endophytic actinomycetes isolated from rice blast infected and healthy rice, Streptomyces endus OsiSh-2, exhibited a remarkable in vitro antagonistic activity. Scanning electron microscopy observation of M.oryzae treated by OsiSh-2 revealed significant morphological alterations in hyphae. In two-year's field tests, spraying OsiSh-2 spore solution (10(7) spores mL(-1) ) is capable of reducing rice blast disease severity by 59.64%. In addition, both fermentation broth of OsiSh-2 and its cell-free filtrates could inhibit growth of M.oryzae, suggesting the presence of active enzymes and secondary metabolites. OsiSh-2 is tested positive for PKS-I and NRPS genes and can produce cellulase, protease, gelatinase, siderophore, IAA and ACC deaminase. Preliminary separation indicated that the methanol extract of OsiSh-2 could suppress growth of pathogen; its major active component was identified as nigericin. Conclusion: Endophytic Streptomyces endus OsiSh-2 has potential as a biocontrol agent against rice blast in agriculture.

[24]

DEFEZ

, ANDREOZZI

, BIANCO

. The overproduction of indole-3-acetic acid (IAA) in endophytes upregulates nitrogen fixation in both bacterial cultures and inoculated rice plants
Microbial Ecology, 2017,74(2):441-452.

DOI:10.1007/s00248-017-0948-4 URL [本文引用: 1]

[25]

JHA

, SUBRAMANIAN R

. Endophytic Pseudomonas pseudoalcaligenes shows better response against the Magnaporthe oryzae than a rhizospheric Bacillus pumilus in Oryza sativa(rice)
Archives of Phytopathology and Plant Protection, 2011,44(6):592-604.

DOI:10.1080/03235400903145400 URL [本文引用: 1]

[26]

ZHU X

, HU Y

, CHEN

, WANG Y

, FANG W

, LI X

. Endophytic fungi from Camellia sinensis show an antimicrobial activity against the rice blast pathogen Magnaporthe grisea.
International Journal of Experimental Botany, 2014,83:57-63.

[本文引用: 1]

[27]

李永刚, 宋兴舜, 赵雪莹, 马凤鸣 . 生防枯草芽孢杆菌L1特性的初步研究
植物保护, 2008,34(1):57-61.

[本文引用: 1]

LI Y

, SONG X

, ZHAO X

, MA F

. Preliminary characterization of Bacillus subtilis strain L1.
Plant Protection, 2008,34(1):57-61. (in Chinese)

[本文引用: 1]

[28]

王光华 , RAAIJMAKERS J

. 生防细菌产生的拮抗物质及其在生物防治中的作用
应用生态学报, 2004,15(6):1100-1104.

[本文引用: 1]

WANG G

, RAAIJMAKERS J

. Antibiotics production by bacterial agents and its role in biological control
Chinese Journal of Applied Ecology, 2004,15(6):1100-1104. (in Chinese)

[本文引用: 1]

[29]

PALAZZINI J

, DUNLAP C

, BOWMAN M

, CHULZE S

. Bacillus velezensis RC 218 as a biocontrol agent to reduce Fusarium head blight and deoxynivalenol accumulation: Genome sequencing and secondary metabolite cluster profiles
Microbiological Research, 2016,192:30-36.

DOI:10.1016/j.micres.2016.06.002 URL [本文引用: 1]

[30]

宗英, 赵月菊, 刘阳, 杨庆利 . 一株贝莱斯芽孢杆菌抑制禾谷镰刀菌的研究
核农学报, 2018,32(2):310-317.

DOI:10.11869/j.issn.100-8551.2018.02.0310 URL [本文引用: 1]

为丰富生物防治菌株资源,本试验从感染赤霉病的麦穗上通过平板对峙法筛选到1株可高效抑制禾谷镰刀菌的细菌JS25R,在温室条件下该菌能有效降低小麦赤霉病的发病率、发病程度和病情指数。根据形态特征、生理生化特性和16S rDNA序列分析,将这株拮抗菌鉴定为贝莱斯芽孢杆菌(Bacillus velezensis)。进一步通过发酵液和挥发性物质抑制禾谷镰刀菌效果分析初步研究该菌抑制禾谷镰刀菌的机理。结果表明,JS25R的发酵液可抑制禾谷镰刀菌的生长,抑菌带宽度为0.58 cm。通过PCR扩增,确定该菌具有生防相关的5个脂肽合成基因。同时,JS25R可产生挥发性物质抑制谷镰刀菌的菌体生长(抑制率为27.7%)和孢子萌发(抑制率为26.7%),禾烷酮、2-壬酮和2-壬醇4种物质可完全抑制禾谷镰刀菌的生长。气相色谱-质谱法(GC-MS)分析结果表明,该菌可产生36种挥发性化合物,主要为酮类、烷类、苯类、醇类等,其中二甲基二硫醚、2-十一。本研究结果为禾谷镰刀菌生物防治提供了菌种材料。

ZONG

, ZHAO Y

, LIU

, YANG Q

. Study on the inhibitory effect of Bacillus velezensis on Fusarium graminearum
Journal of Nuclear Agricultural Sciences, 2018,32(2):310-317. (in Chinese)

DOI:10.11869/j.issn.100-8551.2018.02.0310 URL [本文引用: 1]

孙平平, 崔建潮, 贾晓辉, 王文辉 . 贝莱斯芽孢杆菌L-1 对梨灰霉和青霉病菌的抑制作用评价及全基因组分析
微生物学报, 2018,58(9):1637-1646.

DOI:10.13343/j.cnki.wsxb.20170605 URL [本文引用: 1]

：明确贝莱斯芽孢杆菌（）L-1对梨灰霉和青霉病菌的抑制作用，明确菌株L-1无菌发酵液拮抗活性的稳定性及可能的拮抗机制。通过离体测定、活体测定和病原菌菌丝形态观察，评价菌株L-1对梨灰霉和青霉病菌的拮抗活性。以梨灰霉病菌为供试病原菌，利用牛津杯法测定菌株L-1无菌发酵液拮抗活性的稳定性。利用Pacbio RSⅡ三代测序技术测定L-1的全基因序列，将全基因序列与基因蛋白质序列数据库进行BLAST比对分析，预测菌株L-1可能产生的次生代谢产物及潜在的作用机制。菌株L-1对梨灰霉和青霉病菌的活体抑制率分别为92.88%和77.47%，能引起病原菌菌丝膨大、畸形。菌株L-1在含10% NaCl的培养液中仍能正常生长，其无菌发酵液耐高温、酸、碱、紫外照射和蛋白酶降解，对病原菌具有稳定的拮抗活性。全基因序列分析结果显示菌株L-1有112个基因参与了多种碳源的代谢，可以利用多种碳源进行生长；含有参与亚精胺、海藻糖等与菌株抗逆性相关化合物合成的基因；次生代谢产物预测结果显示：L-1含有合成surfactin、fengycin、bacillibactin、bacillaene、macolactin、difficidin、bacilysin等多种肽聚糖和聚酮糖类抗性化合物的基因簇，以及能够降解病原菌细胞壁的-1，3-葡聚糖酶和几丁质酶相关的基因；此外菌株L-1含有生成乙偶姻等能够诱导植物抗性的基因。菌株L-1能有效拮抗多种梨果采后病害，抗逆性强，拮抗活性稳定，预测菌株L-1能够通过产生多种拮抗活性化合物和细胞壁水解酶类以及诱导植物抗性实现防病效果，具有很大的应用潜力。

SUN P

, CUI J

, JIA X

, WANG W

. Complete genome analysis of Bacillus velezensis L-1 and its inhibitory effect on pear gray and blue mold.
Acta Microbiologica Sinica, 2018,58(9):1637-1646. (in Chinese)

DOI:10.13343/j.cnki.wsxb.20170605 URL [本文引用: 1]

SHAN H

, ZHAO M

, CHEN D

, CHENG J

, LI

, FENG Z

, MA Z

, AN D

. Biocontrol of rice blast by the phenaminomethylacetic acid producer of Bacillus methylotrophicus strain BC79.
Crop Protection, 2013,44:29-37.

DOI:10.1016/j.cropro.2012.10.012 URL [本文引用: 1]

[33]

SHA Y

, WANG

, LI

. Suppression of Magnaporthe oryzae and interaction between Bacillus subtilis and rice plants in the control of rice blast
SpringerPlus, 2016,5(1):1238.

DOI:10.1186/s40064-016-2858-1 URL [本文引用: 1]

[34]

徐婷, 朱天辉, 李姝江, 谯天敏 . 贝莱斯芽孢杆菌Bacillus velezensis YB15 β-葡聚糖酶的抑菌作用与基因克隆
中国生物防治学报, 2014,30(2):276-281.

Magsci [本文引用: 1]

本文在对峙法验证贝莱斯芽孢杆菌B. velezensis YB15具抑菌作用的基础上，用透明圈法、DNS法研究其产β-葡聚糖酶特性，利用对峙法验证该酶抑菌作用，通过PCR法获得目的基因，分析克隆序列并预测其蛋白质结构与功能。结果表明，该菌株对多种病原真菌有拮抗作用，杨树紫纹羽病菌拮抗带达11.0 mm，该菌提取的葡聚糖酶粗酶液对杨树紫纹羽病菌抑菌带为10.6 mm，说明葡聚糖酶在菌株YB15抑菌中有重要作用。不同接种方法影响菌株YB15葡聚糖酶水解透明圈形成，点种法水解圈与菌落直径之比在72 h可达14.1，效果最好。克隆所得菌株YB15葡聚糖酶基因命名为Bglu1，该基因序列长732 bp，编码243个氨基酸，此酶蛋白氨基酸序列与解淀粉芽孢杆菌TB2 β-1,3-1,4-葡聚糖酶同源性较高，属糖基水解酶16家族，N端疏水区存在信号肽并具跨膜区域，推测其为分泌蛋白。

, ZHU T

, LI S

, QIAO T

. Fungus-inhibitory activity and gene cloning of β-glucanase from Bacillus velezensis YB15
Chinese Journal of Biological Control, 2014,30(2):276-281. (in Chinese)

Magsci [本文引用: 1]

李湘民, 许志刚 MEW T

, . 稻株上拮抗细菌的定殖及其对土著细菌的影响
生态学报, 2008,28(8):3868-3874.

Magsci [本文引用: 1]

在温室条件下，通过分批播种、接种纹枯病菌Rhizoctonia solani，以及在水稻分孽盛期喷雾拮抗细菌Pseudomonas fluorescens Pf7-14 (天然的抗萘啶酮酸菌株) 和B5423-R(Bacillus subtilis B5423的利福平抗性突变体)的菌悬浮液，并通过定期取样，平板系列稀释法回收，测定了菌株Pf7-14、B5423-R和土著细菌群体在水稻健株和纹枯病株上的种群数量，所获结果如下：①当相同的浓度(约2.0×108cfu/ml)的菌悬浮液喷雾到叶片时，菌株Pf7-14定殖的时间比菌株B5423-R长，且在相同的时间内，菌株Pf7-14的平均群体数量高于菌株B5423-R；②在健康的水稻茎部，菌株Pf7-14的两个高、低不同浓度处理的平均群体数量均表现出随时间降低的趋势；相比，较低浓度(4.0×107cfu/ml)的B5423-R在茎部的平均群体数量随着时间的下降，而较高浓度(20×108cfu/ml)的B5423-R的平均群体数量在水稻乳熟至黄熟期保持稳定或略有增长；③当病斑面积占取样茎面积的比率为20%～35%时，在应用1和14d后菌株Pf7-14在健茎的平均群体数量分别是病茎的6倍多和2倍多，差异均达到显著性的水平(P=0.05)，而菌株B5423-R 在应用1d后病茎的数量比在健茎显著性地低大约1倍，但在7～14d后，病茎的数量比在健茎显著性地高5~6倍，群体在病茎表现出相对的增长； ④土著细菌群体在病斑茎部是健茎的6～7倍。这些结果表明两类拮抗细菌有着明显不同的定殖习性，在病斑上B5423比Pf7-14具有更强的竞争能力，是一类更好的生防制剂；同时表明引入的拮抗细菌同土著细菌群体在营养和空间上竞争激烈，且土著细菌群体更具有竞争优势。

LI X

, XU Z

, MEW T

. Colonization of antagonistic bacteria on rice plants and their influence on native bacteria
Acta Ecologica Sinica, 2008,28(8):3868-3874. (in Chinese)

Magsci [本文引用: 1]

赵达, 傅俊范, 裘季燕, 刘伟成 . 枯草芽孢杆菌在植病生防中的作用机制与应用
辽宁农业科学, 2007(1):46-48.

[本文引用: 1]

ZHAO

, FU J

, QIU J

, LIU W

. Bio-control mechanism and application ofBacillus subtilis in plant disease
Liaoning Agricultural Sciences, 2007(1):46-48. (in Chinese)

[本文引用: 1]

[37]

RENGPIPAT

, WONGTANGPRASERT

, PALAGA

. The use of green fluorescent protein as a marker for monitoring a probiotic Bacillus S11 in the black tiger shrimp Penaeus monodon
Aquaculture Nutrition, 2009,15(3):297-305.

DOI:10.1111/anu.2009.15.issue-3 URL [本文引用: 1]

[38]

PADDA K

, PURI

, ZENG Q

, CHANWAY C

, WU X

. Effect of GFP-tagging on nitrogen fixation and plant growth promotion of an endophytic diazotrophic strain of aenibacillus polymyxa
PBotany, 2017,95(9):933-942.

DOI:10.1139/cjb-2017-0056 URL [本文引用: 1]

Green fluorescent protein (GFP), a renowned marker protein, is typically believed to be inert in affecting the physiology of host bacteria. We analyzed the effects of GFP-tagging on the ability of an endophytic diazotroph, Paenibacillus polymyxa P2b-2R, to fix nitrogen and promote overall growth of corn plants. The growth response and the amount of nitrogen fixed by P2b-2Rgfp-inoculated plants were compared with uninoculated controls and P2b-2R-inoculated plants at three harvests. P2b-2Rgfp inoculation significantly increased the biomass of corn plants as compared to non-inoculated controls and P2b-2R-treated plants. In vitro tests revealed that strains P2b-2R and P2b-2Rgfp possess various plant-growth-promoting characteristics, namely phosphate solubilization, production of siderophores, IAA, ammonia, and enzymes like cellulase, protease, and catalase. P2b-2Rgfp-inoculated plants fixed 18% atmospheric nitrogen, significantly higher than P2b-2R-inoculated plants (15%). This difference led us to compare the expression of structural nif genes (nifH, nifD, nifK) of strains P2b-2R and P2b-2Rgfp. It was observed that expression levels of structural nif genes of strain P2b-2Rgfp were 1.5-fold higher than those of strain P2b-2R. These results indicate that GFP-tagging positively affects the efficacy of strain P2b-2R to promote plant growth and fix nitrogen, perhaps by increasing the expression levels of structural nif genes.

[39]

ZIMMER

. Green fluorescent protein (GFP): application, structure, and related photophysical behavior
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LIU X

, ZHAO H

, CHEN S

. Colonization of maize and rice plants by strain Bacillus megaterium C₄.
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DOI:10.1007/s00284-005-0162-3 URLPMID:16502291 [本文引用: 1]

Bacillus megaterium C4, a nitrogen-fixing bacterium, was marked with the gfp gene. Maize and rice seedlings were inoculated with the, GFP-labeled B. megaterium C4 and then grown in gnotobiotic condition. Observation by confocal laser scanning microscope showed that the GFP-labeled bacterial cells infected the maize roots through the cracks formed at the lateral root junctions and then penetrated into cortex, xylem, and pith, and that the bacteria migrated slowly from roots to stems and leaves. The bacteria were mainly located in the intercellular spaces, although a few bacterial cells were also present within the xylem vessels, root hair cells, epidermis, cortical parenchyma, and pith cells. In addition, microscopic observation also revealed clearly that the root tip in the zone of elongation and differentiation and the junction between the primary and the lateral roots were the two sites for the bacteria entry into rice root. Therefore, we conclude that this Gram-positive nitrogen-fixer has a colonization pattern similar to those of many Gram-negative diazotrophs, such as Azospirillun brasilense Yu62 and Azoarcus sp. As far as we know, this is the first detailed report of the colonization pattern for Gram-positive diazotrophic Bacillus.

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NG L

, SARIAH

, SARIAM

, RADZIAH

, ZAINAL ABIDIN

M A

. Bio-efficacy of microbial-fortified rice straw compost on rice blast disease severity, growth and yield of aerobic rice
Australasian Plant Pathology, 2012,41(5):541-549.

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KANJANAMANEESATHIAN

, CHUMTHONG

, PENGNOO

, WIWATTANAPATAPEE

. Bacillus megaterium suppresses major Thailand rice diseases
Asian Journal of Food and Agro-Industry, 2009,2(Special Issue):S154-S159.

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KYUNG-SEOK

, DIBY

, WAN-HAE

. Bacillus vallismortis EXTN-1-mediated growth promotion and disease suppression in rice
The Plant Pathology Journal, 2006,22(3):278-282.

DOI:10.5423/PPJ.2006.22.3.278 URL [本文引用: 1]