烟草赤星病菌拮抗性大豆根瘤内生菌的筛选及抑制作用

doi:10.13287/j.1001-9332.201605.024

摘要/Abstract

摘要： 以烟草赤星病菌为供试病原菌,采用对峙法、代谢液培养法对分离自大豆根瘤的内生细菌进行抑菌筛选,对筛选菌株作用的病原菌菌丝进行显微观察,研究筛选菌株细胞培养特征及生理生化特性、16S rDNA测序序列、系统发育,以及进行温室接种防病试验.结果表明: 大豆根瘤内生菌经初筛、复筛,对烟草赤星病菌抑菌率42%以上的有7株菌,分别属于芽孢杆菌属、假单胞菌属、中华根瘤菌属和寡养单胞菌属.显微观察表明,在拮抗性内生菌作用下病原菌菌丝末端出现明显畸形,呈珊瑚状分枝、球状膨大等;代谢液试验表明,内生菌对病原菌的抑制作用主要是内生菌代谢产生的胞外物质在发挥作用;对峙试验表明,芽孢杆菌可迅速形成生物薄膜,有效阻碍病原菌丝生长和延伸.盆栽防病试验表明,拮抗性内生菌处理下烟草病情指数显著低于对照,表明筛选菌株可作为烟草赤星病的生防菌株资源.

关键词: 根瘤内生菌, 烟草赤星病菌, 系统发育, 16SrDNA测序, 生物防治

Abstract: Using Alternaria longipes as tested phytopathogen, endophytic bacteria isolated from soybean nodules were selected to study antagonistic effects by confrontation and metabolic liquid culture methods. The inhibited hyphae were determined by microscopic observation, and the screened strains were characterized by cell culture, physiological and biochemical tests, 16S rDNA sequencing, phylogenetic analysis and inoculation trials in greenhouse. The results indicated that the seven of the endophytes exerted more than 42% inhibitory effects after the first and the second screening. These strains belonged to genus Bacillus, Pseudomonas, Sinorhizobium and Stenotrophomonas, respectively. Microscopic observation showed that the affected hyphae ends of A. longipes appear deformity of coralline branch, spherical expansions and so on. Antagonistic experiments with metabolites showed that the inhibition of endophytic bacteria against pathogenic fungus played an effective role mainly by bacteria producing extracellular substances. Confrontation tests suggested that endophytic Bacillus rapidly produced biofilm to effectively hinder the growth and extension of pathogen hyphae. Inoculation experiments showed that the disease index of treatment group was significantly lowered compared with the control, suggesting it could be utilized as a biological control resource inhibiting tobacco brown spot.

Key words: Alternaria longipes, phylogenetic analysis, 16S rDNA sequencing, biocontrol., endophytic bacteria of root nodules

赵龙飞, 徐亚军, 侯怡婷, 邹艳慧, 李亚楠, 杨志华, 李小雨, 张梦瑶. 烟草赤星病菌拮抗性大豆根瘤内生菌的筛选及抑制作用[J]. 应用生态学报, 2016, 27(5): 1560-1568.

ZHAO Long-fei, XU Ya-jun, HOU Yi-ting, ZOU Yan-hui, LI Ya-nan, YANG Zhi-hua, LI Xiao-yu, ZHANG Meng-yao. Screening and inhibition of antagonistic endophytic bacteria isolated from soybean (Glycine max)nodules against Alternaria longipes.[J]. Chinese Journal of Applied Ecology, 2016, 27(5): 1560-1568.

参考文献

[1] Zhao LF, Deng ZS, Yang WQ, et al. Diverse rhizobia associated with Sophora alopecuroides grown in different regions of Loess Plateau in China. Systematic and Applied Microbiology, 2010, 33: 468-477
[2] Shi J-Y (石晶盈), Chen W-X (陈维信), Liu A-Y (刘爱媛). Advances in the study of endophytes and their effects on control of plant diseases. Acta Ecologica Sinica (生态学报), 2006, 26(7): 2395-2401 (in Chinese)
[3] Zhao LF, Xu YJ, Sun R, et al. Identification and cha-racterization of the endophytic plant growth prompter Bacillus cereus strain MQ23 isolated from Sophora alopecuroides root nodules. Brazilian Journal of Microbiology, 2011, 42: 567-575
[4] Kuklinsky-Sobral J, Araújo WL, Mendes R, et al. Isolation and characterization of soybean-associated bacteria and their potential for plant growth promotion. Environmental Microbiology, 2004, 6: 1244-1251
[5] Taghavi S, Barac T, Greenberg B, et al. Horizontal gene transfer to endogenous endophytic bacteria from poplar improves phytoremediation of toluene. Applied and Environmental Microbiology, 2005, 71: 8500-8505
[6] Zhao L-F (赵龙飞). Research on resource diversity of soybean (Glycine max (L.) Merr) nodule endophytes in China. Guangdong Agricultural Sciences (广东农业科学), 2014(9): 15-19 (in Chinese)
[7] Zhao L-F (赵龙飞), Xu Y-J (徐亚军), Cao D-J (曹冬建), et al. Screening, resistance, phylogeny and growth promoting of phosphorus solubilizing bacteria isolated from soybean root nodules. Acta Ecologica Sinica (生态学报), 2015, 35(13): 4425-4435 (in Chinese)
[8] Xu Y-J (徐亚军), Zhao L-F (赵龙飞), Chen P (陈普), et al. Isolation, screening and characterization of phytopathogen antagonistic endophytes from wild Artemisia argyi. Acta Ecologica Sinica (生态学报), 2013, 33(12): 3697-3705 (in Chinese)
[9] Ma Y, Mani R, Yong ML, et al. Inoculation of endophytic bacteria on host and non-host plants effects on plant growth and Ni uptake. Journal of Hazardous Materials, 2011, 195: 230-237
[10] Miller KI, Qing C, Sze MY, et al. Culturable endophytes of medicinal plants and the genetic basis for their bioactivity. Microbial Ecology, 2012, 64: 431-449
[11] Garrity GM, Bell JA, Lilburn TG. Bergey’s Manual of Systematic Bacteriology, New York: Springer Verlag, 2004
[12] Ma G-H (马冠华), Zhou C-Y (周常勇), Xiao C-G (肖崇刚). Identification of the endophytic bacterial isolate Itb57 from tobacco plant and its control efficacy on tobacco black shank caused by Phytophthora nicotiannae. Acta Phytophylacica Sinica(植物保护学报), 2010, 37(2): 148-152 (in Chinese)
[13] Whipps JM. Microbial interactions and biocontrol in the rhizosphere. Journal of Experimental Botany, 2003, 56: 487-451
[14] Zhao L-F (赵龙飞), Xu Y-J (徐亚军), Lai X-H (赖心河), et al. Screening probiotic endophytic bacteria from medicinal plant Ilex cornuta and the phytopathogen-inhibiting effect. Chinese Journal of Applied Ecology (应用生态学报), 2015, 26(5): 1553-1562 (in Chinese)
[15] Xu Y-J (徐亚军), Zhao L-F (赵龙飞). Study on inhibitory activities of the extract from field Artemisia argyi to Escherichia coli. Jiangsu Agricultural Sciences (江苏农业科学), 2012, 40(4): 306-308 (in Chinese)
[16] Compant S, Duffy B, Nowak J, et al. Use of plant growth-promoting bacteria for biocontrol of plant diseases: Principles, mechanisms of action, and future prospects. Applied and Environmental Microbiology, 2005, 71: 4951-4959
[17] Yi L (易龙), Xiao C-G (肖崇刚), Ma G-H (马冠华), et al. Inhibition and screening of beneficial endophytic bacteria to control tobacco brown spot. Acta Microbiologica Sinica (微生物学报), 2004, 4(1): 19-22 (in Chinese)
[18] Yi L (易龙), Xiao C-G (肖崇刚). Screening of antagonistic bacteria for biocontrol against tobacco brown spot. Acta Phytophylacica Sinica (植物保护学报), 2004, 31(1): 63-68 (in Chinese)
[19] Lim HS, Kim YS, Kim SD. Pseudomonas stutzeri YPL-1 genetic transformation and antifungal mechanism against Fusarium solanian agent of plant root rot. Applied and Environmental Microbiology, 1991, 57: 510-516
[20] Viviana Y, Houda Z, Inmaculada V, et al. Biological control of peach brown rot (Monilinia spp.) by Bacillus subtilis CPA-8 is based on production of fengycin-like lipopeptides. European Journal of Plant Pathology, 2012, 132: 609-619
[21] Gao L-L (高玲玲), Chen X-L (陈小龙), Jiang T (蒋涛), et al. Isolation and identification of endophytic nitrogen-fixing bacteria in rice with antipathogenic functions. Journal of Huazhong Agricultural University (华中农业大学学报), 2012, 31(5): 553-557 (in Chinese)
[22] Yi L (易龙), Ma G-H (马冠华), Yang S-Y (杨水英), et al. Identification of the antagonistic bacterium Ata 28 and its inhibition of tobacco brown spot. Journal of Southwest University (Natural Science) (西南大学学报: 自然科学版), 2007, 29(3): 100-103 (in Chinese)
[23] Wang J (王静), Tian H (田华), Kong F-Y (孔凡玉), et al. Inhibition of Bacillus pumilus AR03 on Alternaria alternata and Erysiphe cichoracearum on tobacco. Chinese Journal of Applied Ecology (应用生态学报), 2015, 26(10): 3167-3173 (in Chinese)
[24] Li H-L (李洪林). Screening of Pseudomonas fluorescens and Biocontrol of Alternaria alternata. Master Thesis. Daqing: Heilongjiang Bayi Agricultural University, 2008 (in Chinese)
[25] Zhang Z, Yuen GY. The Role of Chitinase Production by Stenotrophomonas maltophilia strain C3 in biological control of Bipolaris sorokiniana. Phytopathology, 2000, 90: 384-389