玉米根际细菌群落特征及生防菌筛选

doi:10.16409/j.cnki.2095-039x.2018.05.017

摘要/Abstract

摘要： 为了明确玉米根际细菌群落结构特征并从中筛选玉米病害生防资源，本研究采用培养法从玉米根际分离得到1584株细菌，分别属于变形菌门、厚壁菌门、放线菌门和拟杆菌门，其中变形菌门为优势菌群。在属水平上，假单胞菌为优势菌群。以玉米大斑菌为靶标菌，通过平板对峙方法，筛选出162株具有抑菌效果的菌株，其中1株抑菌效果最强，根据形态学和分子生物学结合的方法，将其鉴定为解淀粉芽胞杆菌。该菌对4种玉米病害病原菌具有广谱的抑菌效果，且含有脂肽类抗生素合成基因，其脂肽类物质粗提液对病原菌的抑制率高达81.56%。总体来看，该菌株具有较大的生防潜力，可作为生防菌防治玉米病害。

关键词: 根际微生物, 可培养细菌, 解淀粉芽胞杆菌, 生物防治, 玉米

Abstract: To clarify composition bacterial community in maize rhizosphere and screen biocontrol resources for maize diseases, a total of 1584 bacteria strains were isolated from maize rhizosphere by culture-dependent method. The bacteria were classified into Proteobacteria, Actinobacteria, Firmicutes and Bacteroidetes, among which Proteobacteria were the dominant groups. At genus level, Pseudomonas was the most abundant groups. A total of 162 bacteria strains with antagonism against Setosphearia turcica were selected through plate confrontation method and strain D3 had the strongest antifungal effect. Moreover, strain D3 was identified as Bacillus amyloliquefaciens by morphological and molecular methods. Strain D3 had broad-spectrum antifungal effect on maize fungal pathogens, including S. turcica, Colletotrichum graminicola, Fusarium avenaceum and Curvularia lunata. Using the specific primers of lipopeptide gene, the related genes could be amplified by PCR in strain D3. Additionally, cell-free filtrate and the crude extract of lipopeptide from strain D3 had biocontrol effect on pathogen with the inhibition rate of 81.56%. In conclusion, strain D3 can be used as a new biocontrol microorganism against maize fungal diseases.

Key words: rhizosphere microbes, culturable bacteria, Bacillus amyloliquefaciens, biological control, maize

中图分类号:

S476

刘泉成, 张茜茜, 田雪亮, 刘文德. 玉米根际细菌群落特征及生防菌筛选[J]. 中国生物防治学报, 2018, 34(5): 771-778.

LIU Quancheng, ZHANG Xixi, TIAN Xueliang, LIU Wende. Composition of Bacterial Community in Maize Rhizosphere and Screening of Biocontrol Bacteria Strains[J]. journal1, 2018, 34(5): 771-778.

参考文献

[1] Müller D B, Vogel C, Bai Y, et al. The plant microbiota:systems-level insights and perspectives[J]. Annual Review of Genetic, 2016, 50:211-234.
[2] Philippot L, Raaijmakers J M, Lemanceau P, et al. Going back to the roots:the microbial ecology of the rhizosphere[J]. Nature Reviews Microbiology, 2013, 11(11):789.
[3] Berendsen R L, Pieterse C M J, Bakker P A H M. The rhizosphere microbiome and plant health[J]. Trends in Plant Science, 2012, 17(8):478-486.
[4] Mendes R, Kruijt M, de Bruijn I, et al. Deciphering the rhizosphere microbiome for disease-suppressive bacteria[J]. Science, 2011, 332(6033):1097-1100.
[5] Pham V H T, Kim J. Cultivation of unculturable soil bacteria[J]. Trends in Biotechnology, 2012, 30(9):475-484.
[6] Lagier J C, Armougom F, Million M, et al. Microbial culturomics:paradigm shift in the human gut microbiome study[J]. Clinical Microbiology and Infection, 2012, 18(12):1185-1193.
[7] Bai Y, Müller D B, Srinivas G, et al. Functional overlap of the Arabidopsis leaf and root microbiota[J]. Nature, 2015, 528(7582):364.
[8] 张善文, 张传雷. 基于局部判别映射算法的玉米病害识别方法[J]. 农业工程学报, 2014, 30(11):167-172.
[9] 李文英, 彭智平, 杨少海, 等. 植物根际促生菌对香蕉幼苗生长及抗枯萎病效应研究[J]. 园艺学报, 2012, 39(2):234-242.
[10] 孙广正, 姚拓, 刘婷, 等. 植物根际促生菌对3种土传真菌病害病原的抑制作用[J]. 微生物学通报, 2014, 41(11):2293-2300.
[11] 朱飞舟, 陈利玉, 陈汉春. 16S rRNA基因序列分析法鉴定病原细菌[J]. 中南大学学报(医学版), 2013, 38(10):1035-1041.
[12] Yamamoto S, Harayama S. PCR amplification and direct sequencing of gyrB genes with universal primers and their application to the detection and taxonomic analysis of Pseudomonas putida strains[J]. Applied Environmental Microbiology, 1995, 61:1104-1109.
[13] 邓建良, 刘红彦, 刘玉霞, 等. 解淀粉芽孢杆菌YN-1抑制植物病原真菌活性物质鉴定[J]. 植物病理学报, 2010, 40(2):202-209.
[14] Rastogi G, Coaker G L, Leveau J H J. New insights into the structure and function of phyllosphere microbiota through high-throughput molecular approaches[J]. FEMS Microbiology Letters, 2013, 348(1):1-10.
[15] Lundberg D S, Lebeis S L, Paredes S H, et al. Defining the core Arabidopsis thaliana root microbiome[J]. Nature, 2012, 488(7409):86.
[16] Niu B, Paulson J N, Zheng X, et al. Simplified and representative bacterial community of maize roots[J]. Proceedings of the National Academy of Sciences of the United States of America, 2017, 114(12):2450-2459.
[17] Haney C H, Ausubel F M. Plant microbiome blueprints[J]. Science, 2015, 349(6250):788-789.
[18] Leach J E, Triplett L R, Argueso C T, et al. Communication in the phytobiome[J]. Cell, 2017, 169(4):587-596.
[19] Szoboszlay M, Lambers J, Chappell J, et al. Comparison of root system architecture and rhizosphere microbial communities of Balsas teosinte and domesticated corn cultivars[J]. Soil Biology and Biochemistry, 2015, 80:34-44.
[20] Mendes L W, Raaijmakers J M, de Hollander M, et al. Influence of resistance breeding in common bean on rhizosphere microbiome composition and function[J]. The ISME Journal, 2017, 12(1):212.
[21] van der Heijden M G A, Hartmann M. Networking in the plant microbiome[J]. PLoS Biology, 2016, 14(2):e1002378.
[22] Lisboa M P, Bonatto D, Bizani D, et al. Characterization of a bacteriocin-like substance produced by Bacillus Amyloliquefaciens isolated from the Brazilian Atlantic forest[J]. IntMicrobiology, 2010, 2:111-118.
[23] Chen X H, Koumoutsi A, Scholz R, et al. Comparative analysis of the complete genome sequence of the plant growth-promoting bacterium Bacillus amyloliquefaciens FZB42[J]. Nature biotechnology, 2007, 25(9):1007.
[24] Koumoutsi A, Chen X H, Henne A, et al. Structural and functional characterization of gene clusters directing nonribosomal synthesis of bioactive cyclic lipopeptides in Bacillus amyloliquefaciens strain FZB42[J]. Journal of Bacteriology, 2004, 186(4):1084-1096.
[25] Rahman A, Uddin W, Wenner N G. Induced systemic resistance responses in perennial ryegrass against Magnaporthe oryzae elicited by semi-purified surfactinlipopeptides and live cells of Bacillus amyloliquefaciens[J]. Molecular plant pathology, 2015, 16(6):546-558.
[26] 田雪亮, 张恺, 王国梁, 等. 转录组分析揭示玉米大斑病菌对解淀粉芽孢杆菌胁迫响应机制[J]. 中国科学(生命科学), 2016, 5:15.
[27] Yu G Y, Sinclair J B, Hartman G L, et al. Production of iturin A by Bacillus amyloliquefaciens suppressing Rhizoctonia solani[J]. Soil Biology and Biochemistry, 2002, 34(7):955-963.
[28] Chen X H, Koumoutsi A, Scholz R, et al. Genome analysis of Bacillus amyloliquefaciens FZB42 reveals its potential for biocontrol of plant pathogens[J]. Journal of biotechnology, 2009, 140(1-2):27-37.
[29] Chowdhury S P, Hartmann A, Gao X W, et al. Biocontrol mechanism by root-associated Bacillus amyloliquefaciens FZB42-a review[J]. Frontiers in Microbiology, 2015, 6:780.
[30] Olorunleke F E, Kieu N P, Höfte M. Recent advances in Pseudomonas biocontrol[C]//Bacterial-plant interactions:Advance research and future trends, 2015, 167-198.
[31] Smits T H M, Rezzonico F, Blom J, et al. Draft genome sequence of the commercial biocontrol strain Pantoea agglomerans P10c[J]. Genome Announcements, 2015, 3(6):e01448.
[32] Haggag W M, Timmusk S. Colonization of peanut roots by biofilm-forming Paenibacillus polymyxa initiates biocontrol against crown rot disease[J]. Journal of Applied Microbiology, 2008, 104(4):961-969.
[33] Lugtenberg B, Kamilova F. Plant-growth-promoting rhizobacteria[J]. Annual Review of Microbiology, 2009, 63:541-556.
[34] Manzanera M, Narváez-Reinaldo J J, García-Fontana C, et al. Genome sequence of Arthrobacter koreensis 5J12A, a plant growth-promoting and desiccation-tolerant strain[J]. Genome Announcements, 2015, 3(3):e00648.
[35] Lal S, Chiarini L, Tabacchioni S. New insights in Plant-associated Paenibacillus species:Biocontrol and Plant Growth-promoting Activity[M]//Bacilli and Agrobiotechnology. Springer, 2016, 237-279.
[36] Vargas L K, Volpiano C G, Lisboa B B, et al. Potential of Rhizobia as Plant Growth-promoting Rhizobacteria[M]//Microbes for Legume Improvement. Springer, 2017, 153-174.