【目的】利用环境转录组技术,研究复杂稻田土壤中微生物群落主要生理代谢过程的基因表达水平及其对长期施氮磷钾肥(Mineral nitrogen,phosphorus,and potassium,NPK)的响应规律。【方法】针对中国科学院常熟农田生态系统长期定位试验的NPK施肥处理和不施肥对照处理(Control check,CK)稻田土壤,淹水培养2周后提取土壤微生物总RNA进行高通量转录组测序,利用MG-RAST网络分析平台(Metagenomics Analysis Server)进行活性微生物组成分析、基因功能注释及基因功能分类。【结果】细菌是CK和NPK处理稻田土壤微生物的优势类群,占比高达95%以上,细菌中的活性基因主要源于变形菌门(Proteobacteria,占细菌的50%以上)。同时也检测到古菌、真核生物和病毒等多种微生物的活性基因,而古菌中的活性基因主要源于奇古菌门(Thaumarchaeota,约占古菌的70%)。酸杆菌门(Acidobacteria)在NPK处理土壤中的转录活性显著高于CK处理土壤,而其他的细菌及古菌类群的转录活性在CK和NPK处理土壤间无显著性差异。CK和NPK处理土壤中表达量最高的基因是ABC transporter编码基因,与物质跨膜运输紧密相关。基于COG(Clusters of Orthologous Genes)、Subsystem、KEGG(Kyoto Encyclopedia of Genes and Genomes)3种基因功能分类数据库,发现CK和NPK处理土壤中微生物的主要代谢活动均为能量产生与转化、碳水化合物代谢、蛋白代谢和氨基酸代谢,而最活跃的代谢路径为氧化磷酸化及氨酰-tRNA合成。【结论】淹水状态下CK和NPK处理稻田土壤中的活性微生物组成较为一致,仅Acidobacteria的转录活性在两者间差异较大;在微生物的主要代谢活动方面,CK和NPK处理土壤之间基本一致,均以能量获取与蛋白代谢为主,长期施用无机化肥对复杂土壤微生物群落水平的主要代谢活动影响较小。
A vast number of microorganisms colonize the leaf surface of terrestrial plants,known as the phyllosphere,and these microorganisms are thought to be of critical importance in plant growth and health.However,the taxonomic identities and ecological functions of the microorganisms inhabiting the rice phyllosphere remain poorly understood.Using a massive,parallel pyrosequencing technique,we identified the phyllosphere bacterial taxa of four different rice varieties and investigated the microbial response to elevated CO_2(eCO_2)in a rice field of a free-air CO_2 enrichment(FACE) facility located in Jiangsu Province,China.The results showed that the dominant phylotype,the Enterobacteriaceae family of Gammaproteobacteria,accounted for 70.6%-93.8%of the total bacterial communities in the rice phyllosphere.The dominant phylotype was stimulated by eCO_2,with its relative abundance increasing from 70.6%-75.2%at ambient CO_2(aCO_2) to 86.5%-93.8%at eCO_2 in the phyllosphere of rice varieties IIYou084(TY-084),YangLiangYou6(YLY-6),and ZhenXian96(ZX-96).The rare phylotypes,including the bacterial taxa of Sphingobacteriaceae,Xanthomonadaceae,Oxalobacteraceae,Clostridiaceae,and Pseudomonadaceae,were suppressed and their relative abundance decreased from 13.4%-23.0%at aCO_2 to 1.47%-6.11%at eCO_2.Furthermore,the bacterial diversity indices decreased at eCO_2 in the phyllosphere of the rice varieties TY-084,YLY-6,and ZX-96.In contrast,an opposite response pattern was observed for the rice variety of YangDao8(YD-8).In the phyllosphere of this variety,the relative abundance of the dominant phylotype,Enterobacteriaceae,decreased from 94.1%at aCO_2 to 81.4%at eCO_2,while that of the rare phylotypes increased from 3.37%to 6.59%.In addition,eCO_2 appeared to stimulate bacterial diversity in the rice variety YD-8.Our results suggest that the phyllosphere microbial response to eCO_2 might be relative abundance-dependent in paddy fields.
