长期施肥对稻田土壤细菌、古菌多样性和群落结构的影响

稻田土壤是“迷失碳”的重要吸纳场所之一,也是温室气体(CH4和N2O等)的重要排放源.大气温室气体的动态变化与土壤碳氮转化的微生物过程紧密相关.以湖南桃江国家级稻田肥力变化长期定位试验点为平台,采用PCR-克隆测序和实时荧光定量PCR技术,研究不施肥(CK)、施氮磷钾肥(NPK)和氮磷钾肥+秸秆还田(NPKS)3种长期施肥制度(＞25 a)对稻田土壤细菌和古菌群落结构及数量的影响.细菌和古菌16S rRNA基因文库分析结果表明:稻田土壤细菌主要类群为变形菌、酸杆菌、绿弯菌,而古菌主要为泉古菌和广古菌.长期施肥导致土壤细菌和古菌种群结构产生明显差异,与CK相比,NPK和NPKS处理稻田土壤的变形菌、酸杆菌和泉古菌相对丰度增加.LIBSHUFF软件分析结果也表明,16S rRNA基因文库在CK、NPK及NPKS处理间存在显著差异.3种施肥处理的稻田土壤细菌16S rRNA基因拷贝数为每克干土0.58× 1010～1.06×1010个,古菌为每克干土1.16×106 ～ 1.72×106个.施肥(NPK和NPKS)后,细菌和古菌的多样性和数量增加,且NPKS＞NPK.说明长期施肥显著影响土壤细菌和古菌群落结构、多样性及数量.     

毛竹林集约经营对土壤固碳细菌群落结构和多样性的影响

为揭示毛竹集约经营对土壤固碳细菌的影响,分别采集集约经营时间为0、10、15、20年和25年的毛竹林土壤(0—20 cm和20—40 cm)土壤,应用实时荧光定量PCR、T-RFLP以及cbbL基因文库方法,分析毛竹林长期集约经营过程中土壤固碳细菌丰度和群落结构多样性的变化,通过冗余分析(RDA)探讨影响土壤固碳细菌群落的主要环境因素。结果表明,长期的集约经营显著提高了毛竹林表层和亚表层土壤的养分含量,土壤pH值却明显降低。集约经营毛竹林土壤固碳微生物数量并未表现出与SOC的相关性,而与N素水平的变化显著相关。具体表现为:随着集约经营的进行表层cbbL基因丰度呈先上升(10年)后下降的规律,与氮素水平呈正相关(P0.05);亚表层土壤cbbL基因丰度则呈直线下降的趋势,与C∶N呈正相关(P0.05)。集约经营导致表层和亚表层土壤微生物群落结构改变,表层固碳细菌多样性指数下降。由系统发育分析可知,不可培养固碳细菌占56%比例,土壤中共同的优势种类多为变形菌和放线菌,以兼性自养为主。RDA分析结果表明土壤酸化和养分积累是毛竹林土壤固碳细菌群落和多样性变化的重要原因。     

化肥对稻田土壤细菌多样性及硝化、反硝化功能菌组成的影响

以中国科学院桃源农业生态试验站长期定位施肥试验为平台,采用聚合酶链式反应(polymerase chain reaction,PCR)和DNA序列测定技术分析研究了3种长期施肥制度(对照不施肥-CK,单施氮肥-N,氮磷钾肥-NPK)对土壤细菌群落以及硝化、反硝化微生物种群的影响.通过系统分析细菌16S rDNA、细菌的硝化基因氨单加氧酶(ammonia monooxygenase,amoA)和反硝化基因氧化亚氮还原酶(nitrous oxide reductase,nosZ)等基因文库发现,长期单施氮肥导致细菌16S rDNA和amoA的多样性明显低于CK和NPK处理,而nosZ的多样性与之相反,即单施氮肥处理明显高于CK和NPK处理.LUBSHUFF软件统计分析显示:16S rDNA和amoA基因文库在CK与N,CK与NPK,NPK与N处理间均存在显著性差异.而对于nosZ基因文库,N和NPK与CK处理相比呈现出了显著性差异,N与NPK之间的差异没有达到显著水平.上述结果表明长期施用化肥对水稻土细菌的群落结构及硝化和反硝化细菌组成产生了明显的影响,但这种影响因基因类型而异.     

长期施肥对新疆灰漠土土壤微生物群落结构与功能多样性的影响

以20a新疆国家灰漠土土壤肥力与肥料效益长期定位试验为平台,采用常规培养法,结合Biolog技术对可培养微生物、生理菌群数量和碳源利用进行测定分析,研究撂荒(CK0)、耕作不施肥(CK)、不同化肥(N、NK、NP、PK、NPK)、化肥配施低量高量有机肥(NPKM1和NPKM2)和秸秆还田(NPKS)等10种处理土壤微生物特征,揭示长期施肥对土壤微生物群落结构与功能多样性的影响。结果表明:(1)可培养微生物:与CK处理相比,CK0处理显著提高了细菌、放线菌和真菌的数量(P0.05),NPKS处理微生物数量则显著降低(P0.05);不同化肥处理的细菌(除PK处理外)、放线菌(除PK和N处理外)数量也有所增加,增幅在8.14%—135.70%和15.30%—44.78%之间;真菌数量(除NK处理外)则有一定幅度的降低;NPKM1和NPKM2处理,微生物数量最高,细菌分别增加了162.20%和173.75%,放线菌增加了34.39%和39.37%,真菌增加了63.33%和488.33%;(2)生理菌群:与CK0相比,CK处理显著提高了自生固氮菌和亚硝化细菌数量(P0.05),显著降低了氨化细菌和纤维素分解菌数量(P0.05);与CK相比,NPKM1和NPKM2处理显著提高土壤中与氮素转化有关的生理菌群数量(P0.05),不同化肥处理和NPKS处理的影响不相同,NPK处理显著高于其余处理(P0.05);(3)微生物碳源利用:微生物活性表现为NK、NPKM1、NPKM2N、NPK、CKPK、NPKSCK0、NP;CK0处理3个多样性指数以及NPKM1、NPKM2和NK处理Shannon(H)指数最高,其余施肥处理差异不显著;糖类、氨基酸类、羧酸类和胺类是微生物利用的主要碳源。(4)聚类分析表明,除NP处理外,施氮处理土壤有较为相似的碳源利用,细菌和真菌与养分之间有较好的相关性,可培养微生物和生理菌群与微生物碳源利用的相关性较差。因此,长期不同施肥对新疆灰漠土土壤微生物群落结构和功能多样性产生了显著的影响,长期耕作不施肥降低了土壤微生物群落结构和功能多样性,不同化肥配合施用对微生物群落的影响不同,NPK及NPK配施有机肥可提高土壤微生物多样性。     

不同施肥制度土壤微生物量碳氮变化及细菌群落16S rDNA V3 片段PCR产物的DGGE分析

应用化学分析和变性梯度凝胶电泳（DGGE）技术分离PCR扩增的16S rDNA的方法,研究了不同施肥制度对土壤微生物量碳、氮变化及微生物多样性的影响。结果表明,连续15a长期试验下,土壤微生物量碳（SMB-C）和微生物量氮（SMB-N）的含量大小均为长期撂荒（CK0）土壤高于农田土壤,而在农田土壤中,长期施肥的处理（NPK、NPKM、NPKSt和NPKF）高于长期不施肥处理（CK）,不同的种植制度中,长期复种轮作（NPKF）高于长期复种连作（NPK）;各处理的SMB-C/SOC（土壤有机碳）和SMB-N/TN（全氮）的比值的变化趋势与SMB-C和SMB-N变化一致;从PCR-DGGE分析,长期氮磷钾化肥配施有机肥（NPKM）处理的微生物量碳、氮的含量最高,微生物丰度最高,细菌物种最多,其次为长期撂荒（CK0）,CK处理细菌物种最少。UPGMC聚类分析表明NPK和NPKF处理细菌的群落结构相似,CK和CK0处理细菌的群落结构相似,而NPKM和NPKSt处理细菌的群落结构相似。     

施肥和土壤管理对土壤微生物生物量碳、氮和群落结构的影响

以中国科学院沈阳生态试验站的长期定位试验为平台,研究了不同施肥和土壤管理对潮棕壤微生物生物量碳、氮和群落结构的影响。结果表明,裸地和农田处理的微生物生物量碳、氮较低,但是农田处理下施肥增加了微生物生物量,其中NPK+M效果最明显。DGGE图谱显示,处理间细菌条带分布较相似,其中裸地的细菌多样性最高;长期施肥和土壤管理改变了土壤真菌群落结构,施肥增加了真菌多样性,且有机肥的影响大于化肥;不同处理间氨氧化细菌群落结构差异显著,NPK+M显著增加了氨氧化细菌多样性,且无机肥和有机肥对氨氧化细菌群落影响不同。施肥和土壤管理对细菌影响较小,但显著改变了真菌和氨氧化细菌的群落结构。聚类分析结果显示,土壤管理措施较施肥对细菌、真菌和氨氧化细菌群落的影响更为显著。     

典型淹水稻田土壤微生物群落的基因转录活性及其主要生理代谢过程

【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。     

稻草还田对水稻土固氮基因(nifH)组成结构和多样性的影响

以中国科学院桃源农业生态试验站长期定位施肥试验为平台,选取稻草还田(C)、氮磷钾(NPK)、氮磷钾加稻草还田(NPK+C)和不施肥对照(CK)4个处理,在晚稻的分蘖期、孕穗期和成熟期分别采集土样,利用实时定量PCR (Q-PCR)和末端限制性片段多态性(TRFLP)等分子生物学方法研究长期稻草还田对水稻土含nifH基因固氮微生物群落丰度、组成和多样性的影响.结果表明:与对照相比,稻草还田和单施化肥处理均显著增加nifH基因的丰度(分蘖期除外),NPK+C处理中含nifH基因的微生物数量最高;nifH基因组成也受到长期施肥的影响,其中CK处理nifH基因组成与各施肥处理明显不同,C与NPK处理间nifH基因组成存在一定差异,而NPK与NPK+C处理间无显著差异.长期施肥不会引起含nifH基因微生物群落多样性的显著改变.可见,稻草还田不仅引起nifH基因群落的组成发生变化,而且导致其数量显著增加,因而可增加土壤的固氮能力.     

稻草还田对水稻土固氮基因(nifH)组成结构和多样性的影响

以中国科学院桃源农业生态试验站长期定位施肥试验为平台,选取稻草还田(C)、氮磷钾(NPK)、氮磷钾加稻草还田(NPK+C)和不施肥对照(CK)4个处理,在晚稻的分蘖期、孕穗期和成熟期分别采集土样,利用实时定量PCR(Q-PCR)和末端限制性片段多态性(T-RFLP)等分子生物学方法研究长期稻草还田对水稻土含nifH基因固氮微生物群落丰度、组成和多样性的影响.结果表明：与对照相比,稻草还田和单施化肥处理均显著增加nifH基因的丰度(分蘖期除外),NPK+C处理中含nifH基因的微生物数量最高;nifH基因组成也受到长期施肥的影响,其中CK处理nifH基因组成与各施肥处理明显不同,C与NPK处理间nifH基因组成存在一定差异,而NPK与NPK+C处理间无显著差异.长期施肥不会引起含nifH基因微生物群落多样性的显著改变.可见,稻草还田不仅引起nifH基因群落的组成发生变化,而且导致其数量显著增加,因而可增加土壤的固氮能力.     

施用棉秆炭对新疆连作棉花根际土壤微生物群落结构和功能的影响

以棉秆移除(NPK)和棉秆还田(NPKS)为对照,采用平板计数、Biolog和DGGE等3种方法,研究了棉秆移除基础上施用常量棉秆炭(22.50 t·hm^-2,NPKB-1)和增量棉秆炭(45.00 t·hm^-2,NPKB-2)对新疆连作棉花根际土壤微生物数量、群落功能和结构多样性的影响.结果表明：与NPK和NPKS处理相比,棉秆炭施用显著增加了连作棉田根际土壤中细菌和放线菌数量;NPKB-1处理真菌数量显著高于NPK处理,但增量棉秆炭NPKB-2处理与NPK处理差异不显著;2个棉秆炭处理的真菌数量均低于NPKS处理.棉秆炭处理AWCD值较高,显著提高了微生物丰富度指数,可促进利用糖类、氨基酸类和羧酸类碳源的微生物生长,尤其是利用与根系分泌物相关的酚酸类碳源的微生物.DGGE电泳结果表明,施用棉秆炭(尤其是增量棉秆炭)后,土壤细菌DGGE图谱条带数增多,增加了土壤中芽单胞菌门、酸杆菌门、变形菌门和放线菌门中一些菌群的丰度.UPGMC聚类分析表明,NPKB-2处理明显区别于其他处理,而NPKS、NPK和NPKB-1处理细菌群落结构相似.表明高量施用棉秆炭可显著提高棉花根际土壤微生物多样性,并明显改变土壤细菌群落结构,对连作棉田生态系统健康有改善作用.     

Long-term field fertilization alters the diversity of autotrophic bacteria based on the ribulose-1,5-biphosphate carboxylase/oxygenase (RubisCO) large-subunit genes in paddy soil

Carbon dioxide (CO₂) assimilation by autotrophic bacteria is an important process in the soil carbon cycle with major environmental implications. The long-term impact of fertilizer on CO₂ assimilation in the bacterial community of paddy soils remains poorly understood. To narrow this knowledge gap, the composition and abundance of CO₂-assimilating bacteria were investigated using terminal restriction fragment length polymorphism and quantitative PCR of the cbbL gene [that encodes ribulose-1,5-biphosphate carboxylase/oxygenase (RubisCO)] in paddy soils. Soils from three stations in subtropical China were used. Each station is part of a long-term fertilization experiment with three treatments: no fertilizer (CK), chemical fertilizers (NPK), and NPK combined with rice straw (NPKM). At all of the stations, the cbbL-containing bacterial communities were dominated by facultative autotrophic bacteria such as Rhodopseudomonas palustris, Bradyrhizobium japonicum, and Ralstonia eutropha. The community composition in the fertilized soil (NPK and NPKM) was distinct from that in unfertilized soil (CK). The bacterial cbbL abundance (3–8?×?10⁸ copies g soil^?1) and RubisCO activity (0.40–1.76 nmol CO₂ g soil^?1 min^?1) in paddy soils were significantly positively correlated, and both increased with the addition of fertilizer. Among the measured soil parameters, soil organic carbon and pH were the most significant factors influencing the community composition, abundance, and activity of the cbbL-containing bacteria. These results suggest that long-term fertilization has a strong impact on the activity and community of cbbL-containing bacterial populations in paddy soils, especially when straw is combined with chemical fertilizers.     

Application of targeted metagenomics to explore abundance and diversity of CO2-fixing bacterial community using cbbL gene from the rhizosphere of Arachis hypogaea

Sequestration of CO₂ by autotrophic bacteria is a key process of biogeochemical carbon cycling in soil ecosystem. Rhizosphere is a rich niche of microbial activity and diversity, influenced by change in atmospheric CO₂. Structural changes in rhizosphere composition influence microbial communities and the nutrient cycling. In the present study, the bacterial diversity and population dynamics were established using cbbL and 16S rRNA gene targeted metagenomics approach from the rhizosphere of Arachis hypogaea. A total of 108 cbbL clones were obtained from the rhizospheric soil which revealed predominance of cbbL sequences affiliated to Rhizobium leguminosarum, Bradyrhizobium sp., Sinorhizobium meliloti, Ochrobactrum anthropi and a variety of uncultured cbbL harboring bacteria. The 16S rRNA gene clone library exhibited the dominance of Firmicutes (34.4%), Proteobacteria (18.3%), Actinobacteria (17.2%) and Bacteroidetes (16.1%). About 43% nucleotide sequences of 16S rRNA gene clone library were novel genera which showed < 95% homology with published sequences. Gene copy number of cbbL and 16S rRNA genes, determined by quantitative real‐time PCR (qRT PCR), was 9.38 ± 0.75 × 10⁷ and 5.43 ± 0.79 × 10⁸ (per g dry soil), respectively. The results exhibited bacterial community structure with high bacterial diversity and abundance of CO₂‐fixing bacteria, which can be explored further for their role in carbon cycling, sustainable agriculture and environment management.     

Diversity of Green-Like and Red-Like Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Large-Subunit Genes (cbbL) in Differently Managed Agricultural Soils

A PCR-based approach was developed to detect ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) form I large-subunit genes (cbbL) as a functional marker of autotrophic bacteria that fix carbon dioxide via the Calvin-Benson-Bassham cycle. We constructed two different primer sets, targeting the green-like and red-like phylogenetic groups of cbbL genes. The diversity of these cbbL genes was analyzed by the use of three differently managed agricultural soils from a long-term field experiment. cbbL gene fragments were amplified from extracted soil DNAs, and PCR products were cloned and screened by restriction fragment length polymorphism analysis. Selected unique cbbL clones were sequenced and analyzed phylogenetically. The green-like cbbL sequences revealed a very low level of diversity, being closely related to the cbbL genes of Nitrobacter winogradskyi and Nitrobacter vulgaris. In contrast, the red-like cbbL gene libraries revealed a high level of diversity in the two fertilized soils and less diversity in unfertilized soil. The majority of environmental red-like cbbL genes were only distantly related to already known cbbL sequences and even formed separate clusters. In order to extend the database of available red-like cbbL sequences, we amplified cbbL sequences from bacterial type culture strains and from bacterial isolates obtained from the investigated soils. Bacterial isolates harboring the cbbL gene were analyzed phylogenetically on the basis of their 16S rRNA gene sequences. These analyses revealed that bacterial genera such as Bacillus, Streptomyces, and Arthrobacter harbor red-like cbbL genes which fall into the cbbL gene clusters retrieved from the investigated soils.     

不同施肥处理下水稻根际和非根际土壤中氨基糖积累特征

以水稻长期定位施肥试验土壤为研究对象,选取不施肥(CK)、化肥(NPK)、秸秆还田+化肥(NPKS)、30%有机肥+70%化肥(LOM)和60%有机肥+40%化肥(HOM)5种处理,分析水稻分蘖旺期根际土和非根际土中氨基糖积累特征.结果表明: 与CK和NPK处理相比,长期施用有机物料(NPKS、LOM、HOM)显著增加了水稻根际土和非根际土中有机碳、总氨基糖及其氨基单糖(胞壁酸、氨基葡萄糖和氨基半乳糖)含量.不同施肥处理下3种氨基单糖的积累规律不同,说明不同微生物对施肥处理的响应趋势和强度有所不同.受稻田翻耕等均匀化土壤的农事操作影响,各处理总氨基糖含量在根际土与非根际土间无显著差异.氨基糖碳对土壤有机碳积累的贡献范围为24.0～28.3 mg·g^-1,且以NPKS处理最高,HOM和CK处理最低.真菌氨基葡萄糖/胞壁酸比值范围为24.4～36.6,说明该试验点所有处理的根际土与非根际土中有机质的降解与转化过程以真菌为主导,且与NPK和CK相比,NPKS处理的真菌参与度提高,而施用HOM处理的细菌参与度提高.     

Significant role for microbial autotrophy in the sequestration of soil carbon

Soils were incubated for 80 days in a continuously labeled (14)CO(2) atmosphere to measure the amount of labeled C incorporated into the microbial biomass. Microbial assimilation of (14)C differed between soils and accounted for 0.12% to 0.59% of soil organic carbon (SOC). Assuming a terrestrial area of 1.4 × 10(8) km(2), this represents a potential global sequestration of 0.6 to 4.9 Pg C year(-1). Estimated global C sequestration rates suggest a "missing sink" for carbon of between 2 and 3 Pg C year(-1). To determine whether (14)CO(2) incorporation was mediated by autotrophic microorganisms, the diversity and abundance of CO(2)-fixing bacteria and algae were investigated using clone library sequencing, terminal restriction fragment length polymorphism (T-RFLP), and quantitative PCR (qPCR) of the ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) gene (cbbL). Phylogenetic analysis showed that the dominant cbbL-containing bacteria were Azospirillum lipoferum, Rhodopseudomonas palustris, Bradyrhizobium japonicum, Ralstonia eutropha, and cbbL-containing chromophytic algae of the genera Xanthophyta and Bacillariophyta. Multivariate analyses of T-RFLP profiles revealed significant differences in cbbL-containing microbial communities between soils. Differences in cbbL gene diversity were shown to be correlated with differences in SOC content. Bacterial and algal cbbL gene abundances were between 10(6) and 10(8) and 10(3) to 10(5) copies g(-1) soil, respectively. Bacterial cbbL abundance was shown to be positively correlated with RubisCO activity (r = 0.853; P < 0.05), and both cbbL abundance and RubisCO activity were significantly related to the synthesis rates of [(14)C]SOC (r = 0.967 and 0.946, respectively; P < 0.01). These data offer new insights into the importance of microbial autotrophy in terrestrial C cycling.     

Abundance and Diversity of CO<Subscript>2</Subscript>-fixing Bacteria in Grassland Soils Close to Natural Carbon Dioxide Springs

Gaseous conditions at natural CO₂ springs (mofettes) affect many processes in these unique ecosystems. While the response of plants to extreme and fluctuating CO₂ concentrations ([CO₂]) is relatively well documented, little is known on microbial life in mofette soil. Therefore, it was the aim of this study to investigate the abundance and diversity of CO₂-fixing bacteria in grassland soils in different distances to a natural carbon dioxide spring. Samples of the same soil type were collected from the Stavešinci mofette, a natural CO₂ spring which is known for very pure CO₂ emissions, at different distances from the CO₂ releasing vents, at locations that clearly differed in soil CO₂ efflux (from 12.5 to over 200 μmol CO₂ m⁻² s⁻¹ yearly average). Bulk and rhizospheric soil samples were included into analyses. The microbial response was followed by a molecular analysis of cbbL genes, encoding for the large subunit of RubisCO, a carboxylase which is of crucial importance for C assimilation in chemolitoautotrophic microbes. In all samples analyzed, the “red-like” type of cbbL genes could be detected. In contrast, the “green-like” type of cbbL could not be measured by the applied technique. Surprisingly, a reduction of “red-like” cbbL genes copies was observed in bulk soil and rhizosphere samples from the sites with the highest CO₂ concentrations. Furthermore, the diversity pattern of “red-like” cbbL genes changed depending on the CO₂ regime. This indicates that only a part of the autotrophic CO₂-fixing microbes could adapt to the very high CO₂ concentrations and adverse life conditions that are governed by mofette gaseous regime. Urška Videmšek, Alexandra Hagn, Michael Schloter, and Dominik Vodnik contributed equally to this study.     

Elevated CO2 and nitrate levels increase wheat root-associated bacterial abundance and impact rhizosphere microbial community composition and function

Elevated CO₂ stimulates plant growth and affects quantity and composition of root exudates, followed by response of its microbiome. Three scenarios representing nitrate fertilization regimes: limited (30 ppm), moderate (70 ppm) and excess nitrate (100 ppm) were compared under ambient and elevated CO₂ (eCO₂, 850 ppm) to elucidate their combined effects on root-surface-associated bacterial community abundance, structure and function. Wheat root-surface-associated microbiome structure and function, as well as soil and plant properties, were highly influenced by interactions between CO₂ and nitrate levels. Relative abundance of total bacteria per plant increased at eCO₂ under excess nitrate. Elevated CO₂ significantly influenced the abundance of genes encoding enzymes, transporters and secretion systems. Proteobacteria, the largest taxonomic group in wheat roots (~ 75%), is the most influenced group by eCO₂ under all nitrate levels. Rhizobiales, Burkholderiales and Pseudomonadales are responsible for most of these functional changes. A correlation was observed among the five gene-groups whose abundance was significantly changed (secretion systems, particularly type VI secretion system, biofilm formation, pyruvate, fructose and mannose metabolism). These changes in bacterial abundance and gene functions may be the result of alteration in root exudation at eCO₂, leading to changes in bacteria colonization patterns and influencing their fitness and proliferation.Subject terms: Microbiome, Microbial ecology, Metagenomics, Microbial ecology     

Nitrogen Fertilization Has a Stronger Effect on Soil Nitrogen-Fixing Bacterial Communities than Elevated Atmospheric CO2

Biological nitrogen fixation is the primary supply of N to most ecosystems, yet there is considerable uncertainty about how N-fixing bacteria will respond to global change factors such as increasing atmospheric CO₂ and N deposition. Using the nifH gene as a molecular marker, we studied how the community structure of N-fixing soil bacteria from temperate pine, aspen, and sweet gum stands and a brackish tidal marsh responded to multiyear elevated CO₂ conditions. We also examined how N availability, specifically, N fertilization, interacted with elevated CO₂ to affect these communities in the temperate pine forest. Based on data from Sanger sequencing and quantitative PCR, the soil nifH composition in the three forest systems was dominated by species in the Geobacteraceae and, to a lesser extent, Alphaproteobacteria. The N-fixing-bacterial-community structure was subtly altered after 10 or more years of elevated atmospheric CO₂, and the observed shifts differed in each biome. In the pine forest, N fertilization had a stronger effect on nifH community structure than elevated CO₂ and suppressed the diversity and abundance of N-fixing bacteria under elevated atmospheric CO₂ conditions. These results indicate that N-fixing bacteria have complex, interacting responses that will be important for understanding ecosystem productivity in a changing climate.