解淀粉芽胞杆菌HR-2的分离鉴定及对水稻稻瘟病菌的拮抗效果

doi:10.13560/j.cnki.biotech.bull.1985.2020-0970

摘要/Abstract

摘要：

稻瘟病是一种严重威胁全球粮食安全的水稻真菌病害。本研究采用平板对峙法从湖南岳阳地区筛选出1株对水稻稻瘟病菌具有高效拮抗活性的菌株HR-2。通过形态特征验证、16S rRNA、gyrA和tuf基因序列比对分析,鉴定该菌株为解淀粉芽胞杆菌（Bacillus amyloliquefaciens）。抑菌广谱性测定结果表明菌株HR-2对水稻稻瘟病菌、油菜菌核病菌和番茄青枯病菌均有较强的拮抗效果,抑制率分别为82.34%、72.32%和72.64%。盆栽试验结果显示,菌株HR-2 10倍稀释液和100倍稀释液对水稻稻瘟病的防治效果分别达到63.81%和40.77%,其中HR-2 10倍稀释液的防效与50 mg/L嘧菌酯的防效相当。试验结果表明解淀粉芽胞杆菌菌株HR-2抑菌谱广,尤其对水稻稻瘟病具有显著的防治效果,可为抗稻瘟病生物防治药剂的开发奠定基础。

关键词: 水稻稻瘟病, 解淀粉芽胞杆菌, 分离鉴定, 生物防治

Abstract:

Magnaporthe oryzae is a rice fungal disease that seriously threatens global food security. A strain HR-2 with high antagonistic activity against M. oryzae was screened from Yueyang district,Hunan province by plate confrontation method in this study. The strain HR-2 was identified as Bacillus amyloliquefaciens by morphological verification,16S rRNA,gyrA and tuf gene sequence analysis. The results of broad-spectrum inhibition showed that the strain HR-2 had a fine inhibitory effect on M. oryzae,Sclerotinia sclerotiorum and Ralstonia solanacearum,and the inhibition rates were 82.34%,72.32% and 72.64%,respectively. The results of pot experiment demonstrated that the control effects of HR-2 10-times diluted solution and 100-times diluted solution on M. oryzae were 63.81% and 40.77% respectively,among which the control effects of HR-2 10-times diluted solution were similar to that of 50 mg/L azoxystrobin. The above results indicated that B. amyloliquefaciens HR-2 had a wide antibacterial spectrum and an obvious control effect on M. oryzae. Our research lays a foundation for the development of biological control agent against M. oryzae .

Key words: Magnaporthe oryzae, Bacillus amyloliquefaciens, isolated and identification, biological control

李瑾, 彭可为, 潘求一, 朱哲远, 彭迪. 解淀粉芽胞杆菌HR-2的分离鉴定及对水稻稻瘟病菌的拮抗效果[J]. 生物技术通报, 2021, 37(3): 27-34.

LI Jin, PENG Ke-wei, PAN Qiu-yi, ZHU Zhe-yuan, PENG Di. Isolation and Identification of Bacillus amyloliquefaciens HR-2 and Biological Control of Rice Blast[J]. Biotechnology Bulletin, 2021, 37(3): 27-34.

图/表 11

表1 16S rRNA、gyrA和tuf引物序列

引物名称	引物序列（5'-3'）
27 F	AGAGTTTGATCCTGGCTCAG
1492 R	GGTTACCTTGTTACGACTT
gyrA F	CAGTCAGGAAATGCGTACGTCCTT
gyrA R	CAAGGTAATGCTCCAGGCATTGCT
tuf GPF	ACGTTGACTGCCCAGGACAC
tuf GPR	GATACCAGTTACGTCAGTTGTACGGA

表2 7株菌株对5种供试病原菌的抑菌效果

供试病原菌	HR-2	G-1	G-2	G-3	G-4	G-10	HR-8
水稻稻瘟病菌	82.34±0.08a	82.54±0.04a	82.74±0.06a	83.93±0.06a	82.14±0.18a	81.55±0.16a	82.44±0.04a
油菜菌核病菌	72.32±0.04b	68.25±0.68a	68.06±0.68a	68.65±0.69a	/	/	/
番茄青枯病菌	72.62±0.02a	76.19±0.02a	73.81±0.01a	73.81±0.01a	/	/	/
小麦赤霉病菌	21.05±0.2a	/	13.16±0.05a	31.58±0.1a	/	/	/
西瓜枯萎病菌	26.32±0.16a	/	/	/	/	/	/

图1 菌株HR-2的抑菌活性图片显示菌株HR-2与水稻稻瘟病菌（A）、油菜菌核病菌（B）和番茄青枯病菌（C）对峙培养后病原菌生长情况;对照组（CK）用无菌水处理,实验组（处理）用HR-2菌悬液处理;对照组和实验组接种时间均相同

图2 菌株HR-2菌落形态

图3 菌株HR-2的16S rRNA扩增产物片段

图4 根据16S rRNA基因序列构建菌株HR-2系统发育树分支上的数值表示采用Neighbor-Joining方法构建系统树时,1000次计算时形成该节点的百分比;括号内为菌株的16S rRNA基因序列在GenBank中的登录号;标尺表示碱基置换频率

图5 根据gyrA基因序列构建菌株HR-2系统发育树分支上的数值表示采用Neighbor-Joining方法构建系统树时,1000次计算时形成该节点的百分比;括号内为菌株的gyrA基因序列在GenBank中的登录号;标尺表示碱基置换频率

图6 根据tuf基因序列构建菌株HR-2系统发育树分支上的数值表示采用Neighbor-Joining方法构建系统树时,1000次计算时形成该节点的百分比;括号内为菌株的tuf基因序列在GenBank中的登录号;标尺表示碱基置换频率

图7 解淀粉芽胞杆菌HR-2的生长曲线

表3 四组不同处理对稻瘟病的防治效果

处理	病情指数	防效/%
CK	46.75±0.35a	∕
100 times dilution	27.74±1.07b	40.77±1.91b
10 times dilution	16.96±0.92c	63.81±1.93a
50 mg/L Azoxystrobin	16.18±0.87c	65.48±1.84a

图8 四种不同处理的水稻叶片局部图图片显示水稻叶片处理7-8 d后叶瘟发病状况,根据叶片上的病斑数量评估不同处理对水稻稻瘟病的防治效果。对照组：将无菌水（abc）喷施到水稻叶面上,接种稻瘟孢子悬浮液。处理组：将HR-2-100（def）喷施到水稻叶面上,接种稻瘟孢子悬浮液;将HR-2-10（ghi）喷施到水稻叶面上,接种稻瘟孢子悬浮液;将50 mg/L嘧菌酯（jkl）喷施到水稻叶面上,接种稻瘟孢子悬浮液

参考文献 26

[1]	Miah G, Rafii MY, Ismail MR, et al. Blast disease intimidation towards rice cultivation:a review of pathogen and strategies to control[J]. Journal of Animal and Plant Sciences, 2017,27(4):1058-1066.
[2]	Asibi AE, Qiang C, et al. Rice blast:A disease with implications for global food security[J]. Agronomy, 2019,9(8):451.
[3]	Spence CA, Raman V, Donofrio NM, et al. Global gene expression in rice blast pathogen Magnaporthe oryzae treated with a natural rice soil isolate[J]. Planta, 2014,239(1):171-185.
[4]	Yoon MY, Cha B, Kim JC. Recent trends in studies on botanical fungicides in agriculture[J]. Plant Pathol J, 2013,29(1):1-9.
[5]	Wu CH, Bernard SM, Andersen GL, et al. Developing microbe-plant interactions for applications in plant-growth promotion and disease control, production of useful compounds, remediation and carbon sequestration[J]. Microb Biotechnol, 2009,2(4):428-440.
[6]	Dong YL, Hui L, Rong SH, et al. Isolation and evaluation of Bacillus amyloliquefaciens Rdx5 as a potential biocontrol agent aga Magnaporthe oryzae[J]. Biotechnology & Biotechnological Equipment, 2019,33(1):1-11.
[7]	朱华珺, 周瑚, 任佐华, 等. 枯草芽孢杆菌JN005胞外抗菌物质及对水稻叶瘟防治效果[J]. 中国水稻科学, 2020,34(5):470-480.
	Zhu HJ, Zhou H, Ren ZH, et al. Extracellular antibacterial substance of Bacillus subtilis JN005 and its control effect on rice leaf blast[J]. China Rice Science, 2020,34(5):470-480.
[8]	周瑚, 邹秋霞, 胡玲, 等. 特基拉芽孢杆菌JN-369的分离鉴定及其抑菌物质分析[J]. 农药学学报, 2019,21(1):52-58.
	Zhou H, Zou QX, Hu L, et al. Isolation and identification of Bacillus tequilensis JN-369 and analysis of its antibacterial substances[J]. Journal of Pesticide Science, 2019,21(1):52-58.
[9]	沙月霞, 隋书婷, 曾庆超, 等. 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 YX, Sui ST, Zeng QC, et al. The potential of Bacillus velezensis E69 to prevent rice blast and other fungal diseases[J]. Agricultural Sciences in China, 2019,52(11):1908-1917.
[10]	Huang WX, Liu XY, Zhou XS, et al. Calcium signaling is suppressed in magnaporthe oryzae conidia by Bacillus cereus HS24[J]. Phytopathology, 2020,110(2):1-33.
[11]	王继华, 徐世强, 张木清. 解淀粉芽孢杆菌的研究进展[J]. 亚热带农业研究, 2017,13(3):191-195.
	Wang JH, Xu SQ, Zhang MQ. Research progress of Bacillus amyloliquefaciens[J]. Subtropical Agricultural Research, 2017,13(3):191-195.
[12]	王玉双, 肖蓉, 郭照辉, 等. 来源于中药的稻瘟病菌拮抗解淀粉芽孢杆菌鉴定及其活性成分特性[J]. 中国生物防治学报, 2018,34(5):746-752.
	Wang YS, Xiao R, Guo ZH, et al. Identification of Magnaporthe grisea derived from traditional Chinese medicine against Bacillus amyloliquefaciens and its active ingredients[J]. Chinese Journal of Biological Control, 2018,34(5):746-752.
[13]	樊丽娟, 郭海, 等. 内生解淀粉芽孢杆菌262AG6抑菌物质的初步探究[J]. 西北农业学报, 2020,29(6):912-920.
	Fan LJ, Guo H, et al. Preliminary study on the antibacterial substance of endogenous Bacillus amyloliquefaciens 262AG6[J]. Northwest Agricultural Journal, 2020,29(6):912-920.
[14]	李生樟, 陈颖, 等. 一株拮抗黄单胞菌的贝莱斯芽孢杆菌的分离和鉴定[J]. 微生物学报, 2019,59(10):1969-1983.
	Li SZ, Chen Y, et al. Isolation and identification of a strain of Bacillus velezensis against Xanthomonas[J], Acta Microbiology, 2019,59(10):1969-1983.
[15]	Cheng TC. Bergey’s manual of determinative bacteriology:8th ed[J]. Journal of Invertebrate Pathology, 1975,25(3):395.
[16]	Bavykin SG, Lysov YP, Zakhariev V, et al. Use of 16S rRNA, 23S rRNA, and gyrB gene sequence analysis to determine phylogenetic relationships of Bacillus cereus group microorganisms[J]. Journal of Clinical Microbiology, 2004,42(8):3711-3730.
[17]	Jongsik C, Kyung SB. Phylogenetic analysis of Bacillus subtilis and related taxa based on partial gyrA gene sequences[J]. Antonie van Leeuwenhoek, 2000,78(2):124-126.
[18]	Caamaño-Antelo S, Fernández-No IC, Böhme K, et al. Genetic discrimination of foodborne pathogenic and spoilage Bacillus spp. based on three housekeeping genes[J]. Food Microbiology, 2015,46:290-291.
[19]	陈孝利. 苜蓿内生菌的分离及生防菌株的筛选与鉴定[D]. 北京:中国农业科学院, 2017.
	Chen XL. Isolation of alfalfa endophytes and screening and identification of biocontrol strains[D]. Beijing:Chinese Academy of Agricultural Sciences, 2017: 21-26.
[20]	He N, Wang P, Ma C, et al. Antibacterial mechanism of chelerythrine isolated from root of Toddalia asiatica(Linn)Lam[J]. BioMed Central, 2018,18(1):1-9.
[21]	国家技术监督局. 中华人民共和国国家标准GB/T 15790-2009. 稻瘟病测报调查规范[S]. 2009-03-27.
	National Standard of the People’s Republic of China. GB/T 15790-2009. Specifications for the survey and report of rice blast[S]. 2009-03-27.
[22]	Amruta1 N, Kumar M, Puneeth M, et al. Exploring the potentiality of novel rhizospheric bacterial strains against the rice blast fungus Magnaporthe oryzae[J]. Plant Pathology Journal, 2018,34(2):126-138.
[23]	Omoboye OO, Oni FE, Batool H, et al. Pseudomonas cyclic lipopeptides suppress the rice blast fungus by induced resistance and direct antagonism[J]. Frontiers in Plant Science, 2019,10:1-15. URL pmid: 30723482
[24]	Rong SH, Xu H, Li L, et al. Antifungal activity of endophytic Bacillus safensis B21 and its potential application as a biopesticide to control rice blast[J]. Pesticide Biochemistry and Physiology, 2020,162:69-77.
[25]	黄元慧, 谢鲲鹏, 王朔, 等. 稻瘟病拮抗菌的筛选及抑菌活性的研究[J]. 微生物学杂志, 2016,36(1):30-35.
	Huang YH, Xie KP, Wang S, et al. Screening of antagonistic bacteria of rice blast and study of its antibacterial activity[J]. Journal of Microbiology, 2016,36(1):30-35.
[26]	刘昆昂, 黄亚丽, 贾振华, 等. 拮抗灰霉病菌的芽孢杆菌筛选、鉴定及其代谢产物抑菌效果研究[J]. 华北农学报, 2020,35(3):200-207.
	Liu KA, Huang YL, Jia ZH, et al. Screening and identification of Bacillus against Botrytis cinerea and antibacterial effect of its metabolites[J]. Journal of North China Agriculture, 2020,35(3):200-207.

编辑推荐 0

Metrics

阅读次数

全文

578

HTML			PDF

最新录用	在线预览	正式出版	最新录用	在线预览	正式出版
0	0	26	0	0	552

来源	本网站	其他网站

次数	537	41
比例	93%	7%

摘要

578

最新录用	在线预览	正式出版

0	0	578

	来源	本网站

	次数	578
	比例	100%