烤烟根际土壤微生物对根系酚酸类物质的响应
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摘要
【目的】烟草连作导致化感物质累积,探索化感物质中主要的酚酸类物质对根际土壤微生物的影响,可为克服烟草连作障碍提供理论依据。【方法】采用盆栽试验方法,将前期分离、鉴定出的烟草根系分泌物中主要酚酸类物质苯甲酸和3-苯丙酸接种到土壤中,模拟烟草多年连作土壤。试验设4个处理:对照(T0),向土壤中加入等量灭菌去离子水;添加苯甲酸3μg/kg土(T1);3-苯丙酸8μg/kg土(T2);同时添加苯甲酸3μg/kg土和3-苯丙酸8μg/kg土(T3),每处理5次重复。以MiSeq测序平台对根际土壤微生物进行高通量测序,探索其对根际土壤微生物的影响,同时采用荧光定量PCR法检测土壤中的茄科劳尔氏菌、短短芽孢杆菌、固氮菌、无机磷细菌、硅酸盐细菌等功能微生物及细菌和真菌的数量变化。【结果】T 1、 T 2处理土壤细菌OTUs(Operational Taxonomic Units)数目分别比对照T0降低了21.5%和17.0%,T3处理OTU数量低于T1和T2处理;T2处理土壤中优势微生物种群增多,结构平衡性降低,门上分类构成和微生物群落构成显著不同于对照。主成分分析与聚类分析显示,T2或T3处理土壤微生物聚类关系较近,都与T0处理较远;T3处理土壤中病原菌数量显著提高,拮抗菌、固氮菌、无机磷细菌、硅酸盐细菌、细菌和真菌数量显著减少,且减少幅度大于T1、T2处理。【结论】根系分泌物中主要酚酸类物质苯甲酸和3-苯丙酸均能明显改变根际土壤微生物区系,降低土壤微生群落多样性,显著增加有害微生物数量的同时大大降低有益微生物数量。两种酚类同时存在的危害效果远大于单一酚类。
【Objectives】To investigate the effect of phenolic acids, a kind of allelopathic substance exudated by roots, on the rhizosphere functional microbiota number, in order to present a theoretical basis on overcoming the obstacle caused by continuous tobacco mono-cropping.【Methods】Benzoic acid and 3-phenylpropanoic acid were the two main phenolic acids isolated and identified from tobacco root exudates in previous studies, which were inoculated into soils to simulate the accumulation of tobacco root exudates after many years continuous mono-cropping. The culture experiment had 4 treatments: adding sterilized deionized water(T0), 3 μg/kg soil of benzoic acid(T1), 8 μg/kg soil of 3-phenylpropanoic acid(T2) and both benzoic acid 3μg/kg soil and 3-phenylpropanoic acid 8 μg/kg soil(T3). High-throughput sequencing using MiSeq system was conducted to evaluate the effect of main phenolic acids on soil bacterial microbes. Real-time PCR was employed to detect the population of Rastonia solanacearum, Brevibacillus brevis, N2-fixing bacteria, phosphate solubilizing bacteria, potassium release bacteria, bacteria and fungi.【Results】Compared with T0, the soil microbial operational taxonomic units(OTUs) in T1 and T2 were decreased by 21.5 % or 17.0 %, respectively, and even lower OTU was in T3. In T2 treatment, the dominant soil microorganisms increased, and structure balance was declined, the phylum and community structures were significantly different from those in T0. Both the principal component(PCoA) and cluster analyses showed that the microbe profiles in treatment T2 and T3 were nearer, and both were relatively far from that in T0. In treatment T3, the population of pathogen was significantly increased while those of antagonist B. Brevis, N2-fixing bacteria, phosphate solubilizing bacteria, potassium release bacteria,bacteria and fungi were significantly reduced, and the extent of reduction was greater than that in treatment T1 or T2.【Conclusions】The accumulation of tobacco root exudates benzoic acid and 3-phenylpropanoic acid could affect rhizosphere soil microbiota significantly by decreasing microbial community diversity, increasing pathogen population, and simultaneously reducing beneficial microbial number. The co-existence of the two phenolic acids would lead even worse impaction.
【Objectives】To investigate the effect of phenolic acids, a kind of allelopathic substance exudated by roots, on the rhizosphere functional microbiota number, in order to present a theoretical basis on overcoming the obstacle caused by continuous tobacco mono-cropping.【Methods】Benzoic acid and 3-phenylpropanoic acid were the two main phenolic acids isolated and identified from tobacco root exudates in previous studies, which were inoculated into soils to simulate the accumulation of tobacco root exudates after many years continuous mono-cropping. The culture experiment had 4 treatments: adding sterilized deionized water(T0), 3 μg/kg soil of benzoic acid(T1), 8 μg/kg soil of 3-phenylpropanoic acid(T2) and both benzoic acid 3μg/kg soil and 3-phenylpropanoic acid 8 μg/kg soil(T3). High-throughput sequencing using MiSeq system was conducted to evaluate the effect of main phenolic acids on soil bacterial microbes. Real-time PCR was employed to detect the population of Rastonia solanacearum, Brevibacillus brevis, N2-fixing bacteria, phosphate solubilizing bacteria, potassium release bacteria, bacteria and fungi.【Results】Compared with T0, the soil microbial operational taxonomic units(OTUs) in T1 and T2 were decreased by 21.5 % or 17.0 %, respectively, and even lower OTU was in T3. In T2 treatment, the dominant soil microorganisms increased, and structure balance was declined, the phylum and community structures were significantly different from those in T0. Both the principal component(PCoA) and cluster analyses showed that the microbe profiles in treatment T2 and T3 were nearer, and both were relatively far from that in T0. In treatment T3, the population of pathogen was significantly increased while those of antagonist B. Brevis, N2-fixing bacteria, phosphate solubilizing bacteria, potassium release bacteria,bacteria and fungi were significantly reduced, and the extent of reduction was greater than that in treatment T1 or T2.【Conclusions】The accumulation of tobacco root exudates benzoic acid and 3-phenylpropanoic acid could affect rhizosphere soil microbiota significantly by decreasing microbial community diversity, increasing pathogen population, and simultaneously reducing beneficial microbial number. The co-existence of the two phenolic acids would lead even worse impaction.
引文
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[2]Weir T,Park S W,Vivanco J.Biochemical and physiological mechanisms mediated by allelochemicals[J].Current Opinion in Plant Biology,2004,7:472-479.
[3]贾志红.植烟土壤化感物质、烤烟根系分泌物与细菌群落对连作的响应[D].北京:中国科学院大学博士论文,2010.Jia Z H.Response of allelochemicals,root exudates and bacterial community in tobacoo-growing soil on continuous cropping[D].Beijing:PhD Dissertation of Chinese Academy of Sciences University,2010.
[4]张明艳,张继光,申国明,等.烟田土壤微生物群落结构及功能微生物的研究现状与展望[J].中国农业科技导报,2014,16(5):115-122.Zhang M Y,Zhang J G,Shen G M,et al.Present reseach status and prospects of microbial communities structure and functional microoragnisms in tobacco-planting soil[J].Journal of Agricultural Science and Technology,2014,16(5):115-122.
[5]夏围围,贾仲君.高通量测序和DGGE分析土壤微生物群落的技术评价[J].微生物学报,2014,54(12):1489-1499.Xia W W,Jia Z J.Comparative analysis of soil microbial communities by pyrosequencing and DGGE[J].Acta Microbiologica Sinica,2014,54(12):1489-1499.
[6]Ushio M,Makoto K,Klaminder J,et al.High-throughput sequencing shows inconsistent results with a microscope-based analysis of the soil prokaryotic community[J].Soil Biology and Biochemistry,2014,76(1):53-56.
[7]Luo C,Tsementzi D,Kyrpides N,et al.Direct comparisons of Illumina vs.Roche 454 sequencing technologies on the same microbial community DNA sample[J].PLoS One,2012,7(2):e30087.
[8]Degnan P H and Ochman H.Illumina-based analysis of microbial community diversity[J].Multidisciplinary Journal of Microbial Ecology,2012,6(1):183-194.
[9]赵爽,罗佳,凌宁,等.基因宏阵列和荧光定量PCR方法对西瓜枯萎病害土壤中尖孢镰刀菌的快速检测和定量[J].土壤学报,2010,47(7):704-708.Zhao S,Luo J,Ling N,et al.Quick check and quantification of Fusarium oxysporum in soil with macroarray and real-time PCR method[J].Acta Pedologica Sinica,2010,47(7):704-708.
[10]Leonardo S,Faranco N,and Antonia I.Real-time PCR detection and quantification of soilborne fungal pathogens:the case of Rosellinia necatrix,Phytophthora nicotianae,P.citrophthora,and Verticillium dahliae[J].Phytopathol Mediterr,2004,43:273-280.
[11]张鹏,王小慧,李蕊,等.生物有机肥对田间蔬菜根际土壤中病原菌和功能菌组成的影响[J].土壤学报,2013,50(2):381-387.Zhang P,Wang X H,Li R,et al.Effect of bioorganic fertilizer on pathogenic and functional bacteria composition in rhizospheric soil of field vegetables[J].Acta Pedologica Sinica,2013,50(2):381-387.
[12]赵斌,何绍江.微生物实验[M].北京:科学出版社,2002.Zhao B,He S J.Microbial experiment[M].Beijing:Science Press,2002.
[13]Liu Y X,Li X,Cai K,et al.Identification of benzoic acid and 3-phenylpropanoic acid in tobacco root exudates and their role in the growth of rhizosphere microorganisms[J].Applid Soil Ecology,2015,93:78-87.
[14]Englerbrecht M C.Modification of a semi-selective medium for the isolation and quantification of Pseudomonas solanacearum[J].ACIAR Bacterial Wilt News Letter,1994,10:3-5.
[15]Liu Y X,Shi J X,Feng Y G,et al.Tobacco bacterial wilt can be biologically controlled by the applicaiton of antagonistic strains in combination with organic fertilizer[J].Biology and Fertility of Soils,2013,49:447-464.
[16]Schonfeld J,Heuer H,Van Elsas J D,et al.Specific and sensitive detection of Ralstonia solanacearum in soil on the basis of PCRamplification of fliC fragments[J].Applled and Environmental Microbiol,2003,69(12):7248-7256.
[17]Shida O,Takagi H,Kadowaki K,et al.Proposal for two new genera,Brevibacillus gen.nov.and Aneurinibacillus gen.nov[J].International Journal of Systematic Bacteriology,1996,46(4):939-946.
[18]Wartiainen I,Eriksson T,Zheng W,et al.Variation in the active diazotrophic community in rice paddy-nifH PCR-DGGE analysis of rhizosphere and bulk soil[J].Applied Soil Ecology,2008,39:65-75.
[19]李兴霖,秦平,葛菁萍,等.共培养提高无机磷细菌解无机磷能力及解无机磷基因(pqqE)的克隆[J].中国农学通报,2015,31(23):47-52.Li X L,Qin P,Ge Q P,et al.Enhancement effect of co-culture on the phosphate solubilizing ability of phosphate solubilizing bacteria and the clone of phosphate solubilizing gene pqqE[J].Chinese Agricultural Science Bulletin,2015,31(23):47-52.
[20]吴金光.两种硅酸盐细菌特异性PCR方法的建立及应用[D].杭州:浙江理工大学硕士学位论文,2009.Wu J G.Development and application of specific PCR methods for two silicate bacteria,Paenibacillus mucilaginosus and P.edaphicus[D].Hangzhou:MS Thesis of Zhejiang Sci-Tech University,2009.
[21]李聪智,谭德明,鲁猛厚,等.聚合酶链反应检测细菌16S rRNA基因[J].湖南医科大学学报,1999,(2):136-138.Li C Z,Tan D M,Lu M H,et al.PCR for detection of 16S rRNAgene bacteria[J].Bulletin of Hunan Medical University,1999,(2):136-138.
[22]Das M,Royer T V,Leff L G.Diversity of fungi,bacteria,and actinomycetes on leaves decomposing in a stream[J].Applled and Environmental Microbiology,2007,73(3):756-767.
[23]朱丽霞,章家恩,刘文高.根系分泌物与根际微生物相互作用研究综述[J].生态环境,2003,12(1):102-105.Zhu L X,Zhang J E,Liu W G.Review of studies on interactions between root exudates and rhizospheric microorganisms[J].Ecology and Environment,2003,12(1):102-105.
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