Screening, Identification and Control Efficacy of Antagonistic Bacteria Against Ralstonia solanacearum and Phytophthora parasitica

doi:10.11924/j.issn.1000-6850.casb2021-0375

Abstract

Abstract:

To provide biocontrol microbial resources for the prevention and control of tobacco rhizome diseases, biocontrol strains with good control efficacy on Ralstonia solanacearum and Phytophthora parasitica were screened. The bacteria strains were isolated from the rhizosphere soil of the diseased tobacco plants, and the inhibitory activity was determined by plate face-off method and filter method. The control efficacy of highly active strains was further detected by pot tests. Molecular biological identification of the highly active strains was carried out by 16S rDNA. The promoting and antibacterial activities of the strains were verified by PCR using 9 pairs of specific primers. Two highly active strains LF-1 and LF-2 were obtained and identified as Bacillus subtilis and Bacillus amyloliquefaciens. The results of activity test showed that the inhibition rate of the two strains was 31.16% and 56.98% on R. solanacearum, and 64.44% and 72.44% on P. parasitica, respectively. The corresponding potted experiment showed that the inhibition rate of the two strains was 69.54% and 72.49% on R. solanacearum, and 65.49% and 68.32% on P. parasitica, respectively. The two strains had good control effect on tobacco rhizome diseases and broad application prospect in tobacco disease control.

Key words: Ralstonia solanacearum, Phytophthora parasitica, Bacillus subtilis, Bacillus amyloliquefaciens, control efficacy

CLC Number:

S435.72

PU Yongyu, BAO Lingfeng, HE Xiang, LIU Rui, ZHANG Qing, SHI Zhufeng, HE Yonghong, YANG Peiwen. Screening, Identification and Control Efficacy of Antagonistic Bacteria Against Ralstonia solanacearum and Phytophthora parasitica[J]. Chinese Agricultural Science Bulletin, 2022, 38(7): 116-123.

Figures/Tables 10

References 35

[1]	腾凯, 陈前锋, 周志成, 等. 烟草连作障碍与土壤理化性质及微生物多样性特征的关联[J]. 微生物学通报, 2020,47(9):2848-2856.
[2]	黎妍妍, 王林, 彭五星, 等. 烟草青枯病不同发病阶段根基土壤微生物群落变化趋势分析[J]. 中国烟草科学, 2020,41(5):73-78.
[3]	JIANG G F, WEI Z, XU J, et al. Bacterial wilt in China: history, current status, and future perspectives[J]. Frontiers in plant science, 2017,8:1549-1558. doi: 10.3389/fpls.2017.01549 URL
[4]	WANG H C, CHEN X J, CAI L T, et al. Race distribution and distribution of sensiticities to mefenoxam among isolates of Phytophthora parasitica var. nicotinae in Guizhou province of China[J]. Crop protection, 2013,52:136-140. doi: 10.1016/j.cropro.2013.05.020 URL
[5]	曹坳程, 刘晓漫, 郭美霞, 等. 作物土传病害的危害及防治技术[J]. 植物保护, 2017,43(2):6-16.
[6]	赵荣艳, 杨靖华, 蒋士君. 烟草病害生物防治研究进展[J]. 安徽农业科学, 2006,34(22):5918-5919,5968.
[7]	刘宪臣. 温湿度对烟草青枯病发生的影响及调控技术研究[D]. 重庆:西南大学, 2014.
[8]	千慧敏, 文艺, 赵辉, 等. 烟草黑胫病和根黑腐生防假单胞杆菌的筛选与鉴定[J]. 中国生物防治学报, 2019,35(6):940-948.
[9]	李小杰, 李成, 姚晨虓, 等. 拮抗烟草疫霉菌的木霉菌株筛选鉴定及防病促生作用研究[J]. 中国烟草科学, 2020,41(3):65-70.
[10]	张德锋, 高艳侠, 王亚军, 等. 贝莱斯芽孢杆菌的分类、拮抗功能及其应用研究进展[J]. 微生物学通报, 2020,47(11):3634-3649.
[11]	张洁梅, 张仁军, 姚正平, 等. 烟草青枯病生防菌的筛选及其田间防效评价[J]. 中国农学通报, 2020,36(28):131-136.
[12]	常栋, 顾建国, 贾方方, 等. 烟草黑胫病不同植物源生防菌的筛选及防效测定[J]. 中国烟草学报, 2020,26(3):84-90.
[13]	周向平, 舒翠华, 滕凯, 等. 内生解淀粉芽孢杆菌Xe01的鉴定及其发酵条件优化[J]. 中国烟草科学, 2020,41(6):58-67.
[14]	冯印印, 李斌, 杨洋, 等. 烟草青枯病菌拮抗细菌的筛选,鉴定和抑菌机制研究[J]. 中国生物防治学报, 2021,37(2):9.
[15]	黄瑞环, 芶剑渝, 韩小斌, 等. 烟草主要病害拮抗菌的筛选鉴定及除草活性分析[J]. 烟草科技, 2019,52(12):17-22.
[16]	杨革. 微生物学实验教程[M]. 北京: 科学出版社, 2004:58-123.
[17]	东秀珠, 蔡妙英. 常见细菌系统鉴定手册[M]. 北京: 科学出版社, 2001:370-398.
[18]	JOSHI R, MCSPADDEN G B B. Identification and characterization of novel genetic markers associated with biological control activities in Bacillus subtilis[J]. Phytopathology, 2006,96(2):145-154. doi: 10.1094/PHYTO-96-0145 URL
[19]	MORA I, CABREFIGA J, MONTESINOS E. Antimicrobial peptide genes in Bacillus strains from plant environments[J]. International microbiology, 2011,14(4):213-223.
[20]	张蕾, 徐慧, 朱宝利. 根际微生物与植物再植病的发生发展关系[J]. 微生物学报, 2016,56(8):1234-1241.
[21]	高群, 孟宪志, 于洪飞. 连作障碍原因分析及防治途径研究[J]. 山东农业科学, 2006(3):60-63.
[22]	刘重喜, 宋佳, 王相晶, 等. 植物招募防御微生物与植物保护[J]. 中国科学:生命科学, 2016,46(5):491-497.
[23]	RODRIGO M, MARCO K, IRENE de B, et al. Deciphering the rhizosphere microbiomefor disease-suppressive bacteria[J]. Science, 2011,332:1097-1100. doi: 10.1126/science.1203980 URL
[24]	王亚娇, 栗秋生, 纪莉景, 等. 一株西瓜枯萎病生防菌的鉴定与田间防效[J]. 微生物学通报, 2021(6):1976-1984.
[25]	HUANG J F, WEI Z, TAN S H, et al. The rhizosphere soil of diseased tomato plants as a source for novel microorganisms to control bacterial wilt[J]. Applied soil ecology, 2013,72:79-84. doi: 10.1016/j.apsoil.2013.05.017 URL
[26]	崔晓, 徐艳霞, 刘俊杰, 等. 芽孢杆菌在农业生产中的应用[J]. 土壤与作物, 2019,8(1):32-42.
[27]	GYANESHWAR P, NARESH Kumar G, PAREKH L J, et al. Role of soil microorganisms in improving P nutrition of plants[J]. Plant and soil, 2002,245(1):83-93. doi: 10.1023/A:1020663916259 URL
[28]	OCHOA-LOZA F J, ARTIOLA J F, MAIER R M. Stability constants for the complexation of various metals with a rhamnolipid biosurfactant[J]. Journal of environmental quality, 2001,30(2):479-485. doi: 10.2134/jeq2001.302479x URL
[29]	ILLMER P, SCHINNER F. Solubilization of inorganic calcium phosphates- solubilization mechanisms[J]. Soil biology and biochemistry, 1995,27(3):257-263. doi: 10.1016/0038-0717(94)00190-C URL
[30]	SPERBE J I. Solution of mineral phosphates by soil baceria[J]. Nature, 1957,180:994-995. doi: 10.1038/180994a0 URL
[31]	MOLLA M A Z, CHOWDHURY A A, ISLAM A, et al. Microbial mineralization of organic phosphate in soil[J]. Plant and soil, 1984,78(3):393-399. doi: 10.1007/BF02450372 URL
[32]	张美君, 吴庆, 尹翠, 等. 尖镰孢黄瓜专化型枯萎病菌拮抗菌的筛选、鉴定及培养条件优化[J]. 生物技术通报, 2020,36(9):125-136. doi: 10.13560/j.cnki.biotech.bull.1985.2020-0578
[33]	邓涛, 郭立佳, 杨腊英, 等. 香蕉枯萎病拮抗芽孢杆菌脂肽类抗生素基因引物筛选[J]. 分子植物育种, 2020,18(24):8196-8205.
[34]	薛应钰, 李发康, 李培, 等. 苹果树腐烂病菌拮抗放线菌JPD-1的筛选及鉴定[J]. 植物保护学报, 2020,47(1):134-142.
[35]	张宝俊, 张家榕, 韩巨才, 等. 内生解淀粉芽孢杆菌LP-5抗菌蛋白的分离纯化及特性[J]. 植物保护学报, 2010,37(2):143-147.

物质种类	扩增引物	引物序列	合成基因	参考文献
Surfactin	110 F	GTTCTCGCAGTCCAGCAGAAG	SrfAB	[18]
Surfactin	110 R	GCCGAGCGTATCCGTACCGAG	SrfAB	[18]
YNDJ	147 F	CAGAGCGACAGCAATCACAT	yndJ	[18]
YNDJ	147 R	TGAATTTCGGTCCGCTTATC	yndJ	[18]
Fengycin	FEND F1	CCTGCAGAAGGAGAAGTGAAG	fenD	[18]
Fengycin	FEND R1	TGCTCATCGTCTTCCGTTTC	fenD	[18]
Iturin	ITUC F1	TTCACTTTTGATCTGGCGAT	ituC	[18]
Iturin	ITUC R3	CGTCCGGTACATTTTCAC	ituC	[18]
Bacillomycine	BamC2 F	CTGGAAGAGATGCCGCTTAC	bamC	[19]
Bacillomycine	BamC2 R	AAGAGTGCGTTTTCTTCGGA	bamC	[19]
Ferric	dhbC F	ATGTTGGATCAAAACGTTAT	dhbC	[18]
Ferric	dhbC R	TCATATGTGATCCACGCCCA	dhbC	[18]
Subtilosin	sbo1 F	TCGGTTTGTAAACTTCAACTGC	sboA	[19]
Subtilosin	sbo1 R	GTCCACTAGACAAGCGGCTC	sboA	[19]
Auxin	ysnE F	GGCTGTGAACCTTTGCTATG	ysnE	[18]
Auxin	ysnE R	GCTGTTCGGGTCCTCTTTAT	ysnE	[18]
Vitamins	bioA2 F	TTCCACGGCCATTCCTATAC	bioA	[19]
Vitamins	bioA2 R	TTTGTCCCCTTATCCTGCAC	bioA	[19]

物质种类	扩增引物	引物序列	合成基因	参考文献
Surfactin	110 F	GTTCTCGCAGTCCAGCAGAAG	SrfAB	[18]
Surfactin	110 R	GCCGAGCGTATCCGTACCGAG	SrfAB	[18]
YNDJ	147 F	CAGAGCGACAGCAATCACAT	yndJ	[18]
YNDJ	147 R	TGAATTTCGGTCCGCTTATC	yndJ	[18]
Fengycin	FEND F1	CCTGCAGAAGGAGAAGTGAAG	fenD	[18]
Fengycin	FEND R1	TGCTCATCGTCTTCCGTTTC	fenD	[18]
Iturin	ITUC F1	TTCACTTTTGATCTGGCGAT	ituC	[18]
Iturin	ITUC R3	CGTCCGGTACATTTTCAC	ituC	[18]
Bacillomycine	BamC2 F	CTGGAAGAGATGCCGCTTAC	bamC	[19]
Bacillomycine	BamC2 R	AAGAGTGCGTTTTCTTCGGA	bamC	[19]
Ferric	dhbC F	ATGTTGGATCAAAACGTTAT	dhbC	[18]
Ferric	dhbC R	TCATATGTGATCCACGCCCA	dhbC	[18]
Subtilosin	sbo1 F	TCGGTTTGTAAACTTCAACTGC	sboA	[19]
Subtilosin	sbo1 R	GTCCACTAGACAAGCGGCTC	sboA	[19]
Auxin	ysnE F	GGCTGTGAACCTTTGCTATG	ysnE	[18]
Auxin	ysnE R	GCTGTTCGGGTCCTCTTTAT	ysnE	[18]
Vitamins	bioA2 F	TTCCACGGCCATTCCTATAC	bioA	[19]
Vitamins	bioA2 R	TTTGTCCCCTTATCCTGCAC	bioA	[19]

处理		方法
对照	CK1	无菌水（200 mL/盆）
对照	CK2	无菌水（100 mL/盆）+青枯病菌液（100 mL/盆）或无菌水（100 mL/盆）+黑胫病菌液（100 mL/盆）
LF-1/ LF-2	S1	拮抗菌发酵液（100 mL/盆）+青枯病菌液（100 mL/盆）或拮抗菌发酵液（100 mL/盆）+黑胫病菌液（100 mL/盆）
	S2	10^-1拮抗菌发酵液（100 mL/盆）+青枯病菌液（100 mL/盆）或10^-1拮抗菌发酵液（100 mL/盆）+黑胫病菌液（100 mL/盆）
	S3	10^-2拮抗菌发酵液（100 mL/盆）+青枯病菌液（100 mL/盆）或10^-2拮抗菌发酵液（100 mL/盆）+黑胫病菌液（100 mL/盆）
	S4	10^-3拮抗菌发酵液（100 mL/盆）+青枯病菌液（100 mL/盆）或10^-3拮抗菌发酵液（100 mL/盆）+黑胫病菌液（100 mL/盆）

处理		方法
对照	CK1	无菌水（200 mL/盆）
对照	CK2	无菌水（100 mL/盆）+青枯病菌液（100 mL/盆）或无菌水（100 mL/盆）+黑胫病菌液（100 mL/盆）
LF-1/ LF-2	S1	拮抗菌发酵液（100 mL/盆）+青枯病菌液（100 mL/盆）或拮抗菌发酵液（100 mL/盆）+黑胫病菌液（100 mL/盆）
	S2	10^-1拮抗菌发酵液（100 mL/盆）+青枯病菌液（100 mL/盆）或10^-1拮抗菌发酵液（100 mL/盆）+黑胫病菌液（100 mL/盆）
	S3	10^-2拮抗菌发酵液（100 mL/盆）+青枯病菌液（100 mL/盆）或10^-2拮抗菌发酵液（100 mL/盆）+黑胫病菌液（100 mL/盆）
	S4	10^-3拮抗菌发酵液（100 mL/盆）+青枯病菌液（100 mL/盆）或10^-3拮抗菌发酵液（100 mL/盆）+黑胫病菌液（100 mL/盆）

菌株	烟草青枯病菌		烟草黑胫病菌
菌株	抑制圈直径/cm	抑制率/%	病原菌菌落直径/cm	抑制率/%
LF-1	1.40±0.21b	31.16±0.46b	2.08±0.04a	64.44±1.00b
LF-2	2.56±0.02a	56.98±0.43a	1.74±0.04b	72.44±0.89a