Coupling Analysis Based on High Throughput Sequencing Technology of Soil Bacterial Community and Soil Environmental Factors in Continuous Cropping Tobacco Soil
Abstract：To reveal the relationship of soil bacterial community structure and its response to soil environment under continuous cropping, the Illumina platform Hiseq2500 high-throughput sequencing technique was used to sequence Luohe tobacco continuous cropping tobacco planting soil bacteria 16S rRNA V4 area with different fertilizer treatment, and find out the soil bacteria microbial community composition, diversity and interaction between the soil environment and bacteria with redundancy analysis (RDA). All 25 203 operational taxonomic units (OTUs) and 1 600 239 pieces readings in total were detected in this sequential control condition. Heatmap showed that there were significant differences among the character of bacterial communities in continuous cropping tobacco planting soil, and those differences mainly existed in different treatment or community in different growth period while diversity of species showed less. Alpha index showed that continuous cropping tobacco planting soil bacteria changed during mature period among different growth stages, and each index increased in some extent; the diversity index (Shannon and Simpson index) changed little, bacteria's abundance indexes (ACE and Chao1 index) of flue-cured tobacco raised to a high level after transplanted in 50 and 70 d with mixture of earthworm manure and microbial fertilizer(T3) and the indexes of microbial fertilizer(T2) reached to a peak which occurred after transplanted in 30 and 90 d, which indicated that the effects that different treatments posed on bacterial community showed in the abundance of bacterial community. Principal component analysis (PCA) showed that there was a strong correlation within soil environmental factors, thus based on the strong positive correlation, the original 11 factors could be divided into 4 types of soil environmental factors. Redundancy analysis (RDA) showed that the 5 environmental factors (pH, urease, available phosphorus, protease and amylase ) could represent 4 general system evolved from PCA analysis process, explain the 98.03% of the original 11 soil environment variable and highly represent the level of soil environment system. The pH not only affected the diversity of soil bacterial communities, but also the abundance of soil bacterial communities. There was a strong correlation between amylase and ACE index, the strong relationship also existed in the Chao1 index and amylase. This indicated that the rising of soil carbon level had a positive effect on the growing of soil bacterial abundance, rising soil carbon level also contribute to the activity of bacterial community; and the urease activity was negatively correlated with the abundance and diversity of soil bacterial community, which showed that the elevation in single nitrogen level actually poses a negative effect on the variation of soil bacterial community. The results of this study provide a basis for studying the formation mechanism of continuous cropping obstacles at the microbial level.
[1] 王茂胜，陈彭，薛小平，等..长期连作对烤烟产量和质量的影响[J] 耕作与栽培, 2010,(1);8-9, 46. [2] 晋艳, 杨宇虹, 段玉琪, 等. 烤烟轮作、连作对烟叶产量质量的影响[J]. 西南农业学报, 2004,17(B05);267-271. [3] 郑军辉，叶素芬，喻景权. 蔬菜连作障碍产生原因及生物防治[J]. 中国蔬菜，2004 (3 ): 56-58. [4] Hoitink H A J, Stone A G, Han D Y. Suppression of plant diseases by composts [J]. Hort Science, 1996, 32:184-187 [5] Serra-Wittling C, Houot S, Alabouvette C. Increased soil suppressiveness to Fusarium wilt of flax after addition of mucinipal solid waste compost. [J] Soil Biology and Biocbemistry，1996, 28:1207-1214 [6] Hoitink H A ,J, Boehm M ,J. Biocontrol within the context of soil microbial communities: A substrat-dependent phenomenon [J] Annu Rev Phytopathol, 1999, 37:427-446 [7] 周美荣, 孙振江, 申晓强. 蚯蚓粪的研究及应用[J]. 山西农业科学, 2012,40(8):921-924. [8] 张宝贵. 蚯蚓与微生物的相互作用[J]. 生态学报，1997(5):556-560. [9] 宋长青, 吴金水, 陆雅海, 等.中国土壤微生物学研究10年回顾[J] 地球科学进展，2013, 28 (10) :1087-1105. [10] 蒋婧, 宋明华. 植物与土壤微生物在调控生态系统养分循环中的作用[J]. 植物生态学报, 2010, 34(8): 979-988. [11] Pace NR. Problems with“Procraryote” [J] Journal of Bacteriology 2009，191(7):2008-2010. [12] Rinke C,Schwientek P, Sczyrba A, et al. Insights into the phylogeny and coding potential of microbial dark matter. [J] Nature , 2013,499(7459):431-437 [13] Perkd J. Is $1000 a cost, or a price? Chad Nusbaum-co-director of the Genome Sequencing and Analysis Program at the Broad Institute in Cambridge, MA-wants to know the true cost of sequencing, not just the OTUer pricetag. Photo credit: Len Rubenstein. (Click to enlarge)[J]. BioTechniques, 2013, 54(2):71-74 [14] Mason C E, Elemento O. Faster sequencers, larger datasets, new challenges [J]. Genomebiofogy, 2012,13(3):314 [15] Jan Leps, Petr Smilauer. Multivariate Analysis oI EcologicalData using CANOCO[M]. Cambridge: Cambridge University Press.2003 [16] 鲍士旦.土壤农化分析[M].第3版.北京:中国农业出版社，2000:25-114. [17] 关松荫. 土壤酶及其研究法[M]. 北京:农业出版社，1986. [18] 杨兰芳,曾巧,李海波,等. 紫外分光光度法测定土壤过氧化氢酶活性[J]. 土壤通报, 2011, 42(1): 207-210. [19] Magoc T, Salzberg SL. FLASH:fast length adjustment of short reads to improve genome assemblies[J]. Bioinaformatics, 2011,27(21):2957-2963. [20] J Gregory C , Justin K , Jesse S , et al. QIIME allows analysis of high-throughput community sequencing data.[J] Nature Method, 2010, 7 (5):335-336 [21] Edgar, R.C.Search and clustering orders of magnitude faster than BLAST,[J] Bioinformatics, 2010, 26(19),2460-2461. [22] Wang Q, Garrity MG, Tiedje MJ, et al. Naive. Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy.[J] Applied and Environmental Microbiology, 2007，73 (16):5261 5267. [23] Schloss P D, Gevers D, Westcott S L. Reducing the effects of pcr amplification and sequencing artifacts on 16s rRNA-based studies[J].PLoS One, 2011, 6(12): e27310.. [24] 史志华，朱华德，陈佳，等..小流域土壤水分空间异质性及其与环境因子的关系应用[J]. 生态学报，2012, 23 (4):889-895 [25] 高菊生, 黄晶, 董春华, 等. 长期有机无机肥配施对水稻产量及土壤有效养分影响 [J]. 土壤学报, 2015, 51(2): 314-324. [26] 薛超,黄启为,凌宁,等. 连作土壤微生物区系分析、调控及高通量研究方法[J]. 土壤学报, 2011, 48(3): 612-618. [27] Gomez-Alvarez V，Teal T K, Schmidt T M. Systematic artifacts in metagenomes from complex microbial communities[J].The ISME journal, 2009, 3(11):1314-1317. [28] 楼骏,柳勇,李延. 高通量测序技术在土壤微生物多样性研究中的研究进展[J]. 中国农学通报, 2014, 30(15): 256-260. [29] 胡元森, 吴坤, 李翠香, 等. 黄瓜连作对土壤微生物区系影响Ⅱ-基于DGGE方法对微生物种群的变化分析[J].中国农业科学, 40(10): 2267-2273. [30] 樊晓刚, 金物, 李兆君, 等. 不同施肥和耕作制度下土壤微生物多样性研究进展[J]. 植物营养与肥料学报，2010, 16(3):744-751. [31] 时鹏, 高强, 王淑平, 等. 玉米连作及其施肥对土壤微生物群落功能多样性的影响[[J].生态学报，2010, 30(22):6173-6182. [32] Kandeler E, Luxhel J, Tscherko M, et al. Xylanase, intertase and protease at the soil-litter interface of a loamy sand [J].Soil Biology and Biochemistry, 1999 31 (8):1171-1179 [33] 贾曼莉，郭宏，李会科.渭北生草果园土壤有机碳矿化及其与土壤酶活性的关系[J]. 环境科学，2014,35 (7) : 2777-2784.
