贝莱斯芽孢杆菌E69预防稻瘟病等多种真菌病害的潜力

doi:10.3864/j.issn.0578-1752.2019.11.006

摘要/Abstract

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.

Key words: Bacillus velezensis, rice blast, plant pathogenic fungi, biological control, colonization ability

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.

SHA YueXia,SUI ShuTing,ZENG QingChao,SHEN RuiQing. Biocontrol Potential of Bacillus velezensis Strain E69 Against Rice Blast and Other Fungal Diseases[J]. Scientia Agricultura Sinica, 2019, 52(11): 1908-1917.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2019.11.006

https://www.chinaagrisci.com/CN/Y2019/V52/I11/1908

图/表 6

图1

表1

表2

表3

表4

图2

参考文献 43

[1]	DAGDAS Y F, YOSHINO K, DAGDAS G, RYDER L S, BIELSKA E, STEINBERG G, TALBOT N J . Septin-mediated plant cell invasion by the rice blast fungus,Magnaporthe oryzae. Science, 2012,336(6088):1590-1595. doi: 10.1126/science.1222934
[2]	HUANG J, SI W, DENG Q, LI P, YANG S . Rapid evolution of avirulence genes in rice blast fungus Magnaporthe oryzae. BMC Genetics, 2014,15:45.
[3]	CHAIHARN M, CHUNHALEUCHANON S, LUMYONG S . Screening siderophore producing bacteria as potential biological control agent for fungal rice pathogens in Thailand. World Journal of Microbiology and Biotechnology, 2009,25(11):1919-1928. doi: 10.1007/s11274-009-0090-7
[4]	MENG X K, YU J J, YU M N, YIN X L, LIU Y F . Dry flowable formulations of antagonistic Bacillus subtilis strain T429 by spray drying to control rice blast disease. Biological Control, 2015,85:46-51. doi: 10.1016/j.biocontrol.2015.03.004
[5]	沙月霞, 曾庆超, 王昕, 沈瑞清, 刘浩, 王喜刚 . 防治稻瘟病芽胞杆菌的筛选及效果评价. 中国生物防治学报, 2018,34(3):414-422.
	SHA Y X, ZENG Q C, WANG X, SHEN R Q, LIU H, WANG X G . Screening and control efficiency evaluation ofBacillus against rice blast Magnaporthe oryzae. Chinese Journal of Biological Control, 2018,34(3):414-422. (in Chinese)
[6]	刘诗胤 . 生防菌MF-91对水稻主要病害防治效果、根际微生物多样性及稻米品质的影响[D]. 杭州: 杭州师范大学, 2012.
	LIU S Y . Effects of biocontrol bacteria MF-91 on the control efficiency of main diseases, rhizosphere microbial diversity and rice quality of Oryza sativa[D]. Hangzhou: Hangzhou Normal University, 2012. (in Chinese)
[7]	RAIS A, SHAKEEL M, MALIK K, HAFEEZ F Y, YASMIN H, MUMTAZ S, HASSAN M N . Antagonistic Bacillus spp. reduce blast incidence on rice and increase grain yield under field conditions. Microbiological Research, 2018,208:54-62. doi: 10.1016/j.micres.2018.01.009 pmid: 29551212
[8]	SAIKIA R, GOGOI D K, MAZUMDER S, YADAV A, SARMA R K, BORA T C, GOGOI B K . Brevibacillus laterosporus strain BPM3, a potential biocontrol agent isolated from a natural hot water spring of Assam, India. Microbiological Research, 2011,166(3):216-225. doi: 10.1016/j.micres.2010.03.002
[9]	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.
	SHA Y X, WANG Q, LI Y . Screening and prevention of Bacillus biocontrol against rice blast. Chinese Journal of Biological Control, 2016,32(4):474-484. (in Chinese)
[10]	TAGHAVI S, GARAFOLA C, MONCHY S, NEWMAN L, HOFFMAN A, WEYENS N, BARAC T, VANGRONSVELD J, 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
[11]	PRASANNA R, NAIN L, PANDEY A K, SAXENA A K . 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
[12]	TANG Q, PURI A, PADDA K P, CHANWAY C P . 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
[13]	JI S H, GURURANI M A, CHUN S C . 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
[14]	OWNLEY B H, GWINN K D, VEGA F E . Endophytic fungal entomopathogens with activity against plant pathogens: ecology and evolution. BioControl, 2010,55(1):113-128. doi: 10.1007/s10526-009-9241-x
[15]	LIU Y, BAI F R, LI N, WANG W P, CHENG C . 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
[16]	SHYLLA A, SHIVAPRAKASH M K, SHASHIDHAR H E, VISHWAKARMA P, SUDRADHAR M . Production of phytohormones by endophytic bacteria isolated from aerobic rice. Journal of Pure and Applied Microbiology, 2016,10(3):2127-2133.
[17]	SHAHZAD R, WAQAS M, KHAN A L, AL-HOSNI K, KANG S M, SEO C W, LEE I J . 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
[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
	YANG B, CHEN Y, LI X, REN C G, DAI C 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
[19]	RANGJAROEN C, RERKASEM B, TEAUMROONG N, SUNGTHONG R, LUMYONG S . 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
[20]	PHAM V T K, REDIERS H, GHEQUIRE M G K, NGUYEN H H, DE MOT R, VANDERLEYDEN J, SPAEPEN S . 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
[21]	SHAHZAD R, KHAN A L, BILAL S, WAQAS M, KANG S M, LEE I J . 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
[22]	KLAYRAUNG S, NIAMSUP P, POONNOY P, TOPOONYANONT N . Diversity and control of bacterial contamination of plants propagated in temporary immersion bioreactor system. Acta Horticulturae, 2017,1155:439-446.
[23]	XU T, LI Y, ZENG X D, YANG X L, YANG Y Z, YUAN S S, HU X C, ZENG J R, WANG Z Z, LIU Q, LIU Y Q, LIAO H D, TONG C Y, LIU X M, ZHU Y H . 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 pmid: 27293085
[24]	DEFEZ R, ANDREOZZI A, BIANCO C . 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
[25]	JHA Y, SUBRAMANIAN R B . 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
[26]	ZHU X J, HU Y F, CHEN X, WANG Y H, FANG W P, LI X H . 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.
[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.
	LI Y G, SONG X S, ZHAO X Y, MA F M . Preliminary characterization of Bacillus subtilis strain L1. Plant Protection, 2008,34(1):57-61. (in Chinese)
[28]	王光华 , RAAIJMAKERS J M. 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.
	WANG G H, RAAIJMAKERS J M . Antibiotics production by bacterial agents and its role in biological control. Chinese Journal of Applied Ecology, 2004,15(6):1100-1104. (in Chinese)
[29]	PALAZZINI J M, DUNLAP C A, BOWMAN M J, CHULZE S N . 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
[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
	ZONG Y, ZHAO Y J, LIU Y, YANG Q L . 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
[31]	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
	SUN P P, CUI J C, JIA X H, WANG W H . 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
[32]	SHAN H Y, ZHAO M M, CHEN D X, CHENG J L, LI J, FENG Z Z, MA Z Y, AN D R . 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
[33]	SHA Y X, WANG Q, LI Y . 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
[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.
	XU T, ZHU T H, LI S J, QIAO T M . 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)
[35]	李湘民, 许志刚 MEW T W, . 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.
	LI X M, XU Z G, MEW T W . Colonization of antagonistic bacteria on rice plants and their influence on native bacteria. Acta Ecologica Sinica, 2008,28(8):3868-3874. (in Chinese)
[36]	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.
	ZHAO D, FU J F, QIU J Y, LIU W C . Bio-control mechanism and application ofBacillus subtilis in plant disease. Liaoning Agricultural Sciences, 2007(1):46-48. (in Chinese)
[37]	RENGPIPAT S, WONGTANGPRASERT N, PALAGA T . 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
[38]	PADDA K P, PURI A, ZENG Q W, CHANWAY C P, WU X Q . 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
[39]	ZIMMER M . Green fluorescent protein (GFP): application, structure, and related photophysical behavior. Chemical Reviews, 2002,102(3):759-781. doi: 10.1021/cr010142r
[40]	LIU X M, ZHAO H X, CHEN S F . Colonization of maize and rice plants by strain Bacillus megaterium C₄. Current Microbiology, 2006,52(3):186-190. doi: 10.1007/s00284-005-0162-3 pmid: 16502291
[41]	NG L C, SARIAH M, SARIAM O, RADZIAH O, 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. doi: 10.1007/s13313-012-0145-3
[42]	KANJANAMANEESATHIAN M, CHUMTHONG A, PENGNOO A, WIWATTANAPATAPEE R . Bacillus megaterium suppresses major Thailand rice diseases. Asian Journal of Food and Agro-Industry, 2009,2(Special Issue):S154-S159.
[43]	KYUNG-SEOK P, DIBY P, WAN-HAE Y . 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

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	—

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	—

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	+	++

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	—