长期施肥对黑土农田土壤微生物群落的影响

基于中国科学院海伦农业生态试验站长期定位试验区,应用实时荧光定量PCR(Real-time PCR)和变性梯度凝胶电泳(DGGE)技术研究了无施肥(NF)、单施N、P化肥(NP)以及化肥配施有机猪粪肥(NPM)等3种长期施肥措施对黑土区玉米田土壤微生物群落密度和结构的影响.Real-time PCR方法定量NF、NP及NPM措施土壤细菌群落基因组DNA质量分别为381、1 351和1 773 ng g-1干土,真菌群落基因组DNA质量分别113.3、127.3和20.6 ng g-1干土,真菌与细菌的比率分别为0.31、0.09和0.01,NPM措施显著低于另两种施肥方式(p＜0.05).DGGE方法研究表明,NP和NPM措施不能改善土壤细菌和真菌群落的多样性、均匀性及优势菌优势程度;但主成分分析结果显示NP和NPM措施均可改变土壤细菌和真菌群落的构成,且真菌群落的变化更为显著;聚类分析结果显示NP和NPM措施下细菌群落结构较相近,其相似系数为0.89,真菌群落中NP措施与NF措施相近,相似系数为0.63,高于NP与NPM措施的相似系数0.51.上述结果表明有机猪粪肥的长期施用可以显著降低黑土农田土壤真菌与细菌的比率,且明显地改变土壤细菌和真菌群落的结构.     

不同烤烟种植模式对贵州土壤养分、酶活性及细菌群落结构的影响

贵州是我国第二大烤烟种植省份,为获得更高的经济效益,烟区常采用连作的种植模式。本研究采取大田试验,通过设置4个处理:烤烟连作(T)、白菜-烤烟轮作(C-T)、豌豆-烤烟轮作(P-T)、绿肥-烤烟轮作(GM-T),分析不同烤烟种植模式对贵州土壤养分、酶活性及细菌群落结构的影响。结果表明,烤烟轮作使土壤有机质(SOM)、阳离子交换量(CEC)、全氮(TN)、微生物量碳(MBC)、微生物量氮(MBN)和微生物量磷(MBP)分别提高了0.77%~12.44%、10.78%~38.51%、11.76%~17.65%、10.65%~20.67%、36.58%~160.36%和14.66%~36.21%;土壤中铵态氮(AN)、硝态氮(NN)、有效磷(AP)和速效钾(AK)含量分别降低了1.01%~71.67%、19.29%~24.32%、23.09%~52.21%和6.18%~10.09%。与T处理相比,烤烟轮作提高了土壤脲酶和蔗糖酶活性,提升幅度为12.38%~42.96%和4.00%~22.67%,以GM-T处理效果最佳。同时,烤烟轮作提高了细菌OTU数量,并改变了细菌群落的相对丰度,但未对细菌群落组成产生影响。此外,轮作还增加了土壤中有益细菌类群数量,尤其是杆菌类细菌。冗余分析(RDA)结果显示,MBP是驱动群落变异的主要因子,且与黄杆菌属和褚氏杆菌属呈正相关关系。综上所述,通过烤烟轮作可以均衡土壤养分、改善土壤微环境,以GM-T处理效果最佳。本研究结果可为贵州省烤烟的种植提供理论参考,为解决烤烟连作问题提供技术支撑。     

设施种植模式对土壤细菌多样性及群落结构的影响

为了研究有机和常规设施种植模式及轮作对土壤细菌多样性和群落结构的影响,本研究采用Illumina平台Hiseq 2500高通量测序技术,于2016年6月(作物处于收获期)对北京市顺义区不同设施种植模式(分别为有机设施种植模式和常规设施种植模式下的叶菜连作、茄果连作和叶茄轮作)下土壤细菌进行16S r RNA测序。测序质控后共获得17 278个操作分类单元(operational taxonomic units,OTUs),共计318 851条有效序列。比较不同种植模式和轮作下土壤细菌多样性、细菌群落结构组成、相对丰度及土壤理化性质与细菌群落多样性关系的差异性。结果表明:土壤微生物群落结构在有机和常规设施种植模式下差异明显,有机设施种植土壤细菌多样性高于常规设施种植;有机设施种植下轮作与连作土壤细菌群落结构表现出明显差异,而常规设施种植下,两者没有明显差异;有机种植模式下,轮作土壤细菌群落多样性高于连作土壤;设施种植土壤细菌群落主要属于鞘氨醇单胞菌属(Sphingomonas,5.05%)和芽孢杆菌属(Bacillus,4.84%),相对丰度大于0.5%的共有14个属。有机设施种植土壤含有较多促进植物生长、有机质分解的细菌,常规设施种植土壤中降解化学杀虫剂、防治土壤病害、促进硝化过程的细菌较多。RDA分析结果显示土壤细菌群落主要受全磷、速效磷、有机质的影响。Tumebacillus、Candidatus Solibacter和Acidothermus都是分解有机质、利用碳源的细菌属,与土壤有机质含量呈正相关关系。由此可见,设施条件下,有机和常规种植土壤微生物群落结构的差异性主要源于肥料使用、有害生物防治措施和管理方式的不同。有机设施种植模式下,轮作更有利于发挥其改良土壤营养循环和防治土壤病虫害的作用。上述结果为在微生物水平上研究设施条件下不同种植模式的土壤生态质量差异提供了参考。     

渡良濑游水地土壤微生物群落结构与功能研究

利用BIOLOG和磷脂脂肪酸(PLFA)技术,研究了受足尾铜矿废水影响下的日本西那须地区渡良濑游水地东、西两侧土壤生物学特性、微生物群落结构及功能变化.结果表明:东侧(对照区)土壤全铜含量为45.07mg/kg,有效态铜含量为0.42 mg/kg,同西侧相比,平均降低了19.32%和89.95%.土壤呼吸速率主要受土壤有效态铜、土壤有机质和C/N的影响,随土层深度增加,土壤微生物量氮和溶解性氮都迅速减小,东侧土壤具有较强的氮矿化能力,渡良濑游水地东、西两侧0-5 cm土层土壤的微生物群落生理代谢剖面在培养48 h后出现明显变化;土壤微生物群落的平均颜色变化值(AWCD)随培养时间的延长,趋于平缓增加.在48 h时培养后,西侧位点土壤的AWCD值与东侧相比,分别下降了40%,34%和16%;除B位点样品在20-40 cm土层深度的AWCD略高于其它3个位点外,其余样品的AWCD无明显差异.渡良濑游水地东侧土壤微生物对单一碳源底物的利用能力较高,土壤微生物群落利用碳源的功能多样性也较高.渡良濑游水地西侧区域C位点土壤的格兰仕阳性菌、格兰仕阴性菌、放线菌数量均显著高于东侧A位点,但渡良濑游水地东侧区域土壤3大类群微生物中细菌和放线菌比例更高.在渡良濑游水地自然恢复30多年后,东、西两侧土壤微生物群落结构与功能仍存在较明显的差异.     

盐地碱蓬根际土壤细菌群落结构及其功能

盐地碱蓬作为生物改良盐碱地的理想材料,其根际土壤微生物对土壤改良发挥着重要作用。为了深入探索环渤海滨海盐碱地碱蓬根际土壤细菌群落结构组成及其功能,采用Illumina Misep高通量测序平台对环渤海地区滨海盐碱地盐地碱蓬根际土壤和裸地土壤进行测序。从16个样本中获得有效序列734 792条, 4 285个OTUs,归属于41门、100纲、282目、400科、892属、1 577种。盐地碱蓬根际土壤细菌群落由变形菌门(Proteobacteria)、放线菌门(Actinobacteria)、绿弯曲门(Chloroflexi)、拟杆菌门(Bacteroidetes)、芽单胞菌门(Gemmatimonadetes)、酸杆菌门(Acidobacteria)、厚壁菌门(Firmicutes)、蓝藻细菌门(Cyanobacteria)、髌骨细菌门(Patescibacteria、浮霉菌门(Planctomycetes)组成。Alpha多样性计算结果表明,盐地碱蓬根际土壤细菌群落结构多样性高并与裸地土壤间差异显著;LEfSe(LDAEffectSize)分析发现,盐地碱蓬与裸地差异指示种明显不同。PCoA与相关性Heatmap表明,盐地碱蓬、速效氮、速效钾、速效磷、电导率是影响土壤细菌目类水平群落组成的主要因子。PICRUSt(Phylogenetic InvestigationofCommunitiesbyReconstructionofUnobserved States)分析表明微生物群落在新陈代谢等40个功能方面盐地碱蓬根际土壤比裸地土壤高。本研究表明盐地碱蓬覆盖能够降低土壤盐分,增加土壤养分,对土壤细菌群落多样性及其功能有积极作用。     

镉污染对烤烟根际土壤细菌和真菌群落结构的影响

根际土壤微生物是根际土壤生态系统的重要组成部分,为了明确镉污染条件下烤烟根际土壤微生物群落结构变化,采用全生育期盆栽方法,利用分子生物学PCR-DGGE技术,对不同浓度镉污染下烤烟根际细菌和真菌的群落结构进行了分析,结果表明:(1)镉污染浓度为4 mg kg-1处理的根际土壤细菌群落条带数,明显少于其他处理;随镉污染浓度的增加,一些土壤细菌类群逐渐消失,但特定细菌对高浓度镉污染具有较强的耐受能力;根际细菌可归入8个类群,Bacterium ellin、Acidobacteria酸杆菌和Mycobacterium分枝杆菌属对不同浓度镉污染具有较强的适应能力,其中leptolyngbya瘦鞘丝藻属能适应中等浓度的镉污染,在该浓度下,该微生物可大量繁殖为优势种群。(2)随着镉污染浓度增加,部分特有真菌类群出现,但是在中等镉浓度(4 mg kg-1)处理下,部分真菌类群消失;真菌群落结构可归入4个类群,其中优势和共有类群为Fusarium镰刀菌属和未知菌属,对不同浓度镉污染耐受性较强;Acremonium sclerotigenum硬化支顶孢霉对镉污染反应敏感,Rhizopus根霉菌属嗜好高浓度镉的真菌。     

不同种植年限川党参根际土壤细菌群落结构研究

为了探究生长年限对川党参根际土壤细菌群落结构特征及其多样性的影响,阐明细菌群落变化规律,采用IlluminaMiSeq高通量测序技术对1年生、2年生、3年生和4年生的川党参根际土壤细菌16SrRNA序列进行测序分析。结果表明:(1)生长年限显著影响川党参根际土壤细菌群落多样性,其OTU数、Alpha多样性指数均随生长年限增加呈先升高后降低的趋势;(2)生长年限改变了细菌群落组成,但在门水平上优势菌门(相对丰度>5%)相对稳定,均为变形菌门(Proteobacteria)、未分类细菌门(unclassified_Bacteria)、酸杆菌门(Aci-dobacteria)、放线菌门(Actinobacteria)、拟杆菌门(Bacteroidetes)和绿弯菌门(Chloroflexi),其相对丰度总和占 85.71%～91.39%;在属水平上优势菌群相差较大,仅有全噬菌属(Holophaga)和念珠菌属(Candidatus Udaeo-bacter)是4个年限所共有的优势菌属,革兰氏阴性菌cTPY-13(Gram-negative bacterium cTPY-13)则为2年生独有优势菌属,其余则分别为不同年限有相应共有优势菌属;(3)PCoA分析与UPGMA聚类分析显示,各生长年限间细菌群落差异明显,随生长年限增加,不同组间细菌群落结构差异先增大后逐渐减小,细菌群落在2～3年生时演变较为剧烈;(4)土壤速效养分是影响根际土壤细菌群落的主要环境因子,其程度大小为水解氮(HN)>有效磷(AP)>速效钾(AK),优势菌门中酸杆菌门与AP、AK呈显著负相关(P<0.05),拟杆菌门与AP、AK呈显著正相关(P<0.05),而绿弯菌门与HN呈显著负相关(P<0.05)。综上所述,川党参生长年限延长会影响根际土壤细菌群落结构和多样性,从而改变其根际微生态系统。     

毛乌素沙地人工林恢复对土壤剖面化学性质和细菌群落的影响

明确贫瘠沙地人工林恢复过程中土壤化学性质和微生物群落的动态变化可为风沙区人工林的健康经营和土壤肥力培育提供理论依据。以毛乌素沙地广泛分布的不同年限樟子松林为研究对象,运用高通量扩增子测序和FAPROTAX功能预测技术,解析沙地人工林恢复过程中土壤剖面（0~100 cm）化学性质和细菌群落的分布格局及其变化特征。结果表明：（1）人工林恢复提高了土壤有机碳（SOC）、全氮（TN）及全磷（TP）含量,但降低了土壤有效氮（AN）、有效磷（AP）含量和pH;随土层深度增加,TN含量呈显著下降趋势,而AP含量和pH则相反,TP含量无显著变化。（2）人工林恢复显著改变了变形菌门（Proteobacteria）、酸杆菌门（Acidobacteria）和绿弯菌门（Chloroflexi）的相对丰度,增加了土壤细菌群落基于多度覆盖的物种估计量（ACE指数）;随林分发育,细菌群落的垂直空间变异减小。（3）功能预测表明：人工林恢复降低了土壤细菌对有机物质的分解潜力,造林15年后纤维素分解和芳香族化合物降解过程与未造林样点相比分别下降54.65%和72.18%;但增强了氮的固定及反硝化过程,分别增加99.26%和100.5%。（4）冗余分析和相关性分析表明：SOC和pH是影响细菌群落及其潜在生态功能的重要环境因子。综上可知,沙地人工林恢复虽然增加了土壤碳氮磷总量,但人工林恢复至15年时土壤有效氮磷养分仍显著下降,并显著改变了土壤细菌群落多样性与功能。因此,人为调控措施（如调控凋落物分解与养分释放）对提升干旱和贫瘠生境中沙化土壤养分有效性和微生物多样性与功能具有重要作用,今后在风沙区人工林生态恢复工作中应着重关注林下凋落物的就地留存和腐解。     

菜-菌轮作模式下土壤微生物群落结构及多样性的变化

为明确菜-菌轮作模式对土壤微生物的影响,基于高通量测序技术,对4种轮作模式下的土壤微生物群落结构与多样性进行了研究,结果显示：不同轮作模式下土壤样品中真菌和细菌OUT总数分别是2298和15840条,相较于常规轮作模式A,菜-菌轮作模式B、C、D下真菌的OUT总数、ACE指数、Chao1指数、Shannon指数降低显著,Simpson指数增加明显,但细菌的各参数没有显著的差异;其土壤全氮、碱解氮、速效钾、有机质含量显著高于常规轮作模式A;真菌的优势群落是子囊菌门,相对丰度在66%以上,枝孢属、镰刀菌属两类致病菌群的丰度在菜-菌轮作模式B、C、D中降低明显;细菌的优势群落是变形菌门、放线菌门、绿弯菌门以及酸杆菌门,鞘氨醇单胞菌属、伯克霍尔德氏菌属丰度在菜-菌轮作模式B、C、D中显著提高;聚类分析表明菜-菌轮作模式C、D下微生物群落结构相似度最高,常规轮作模式A可划分为区别于菜-菌轮作模式B、C、D的单独类群。综上所述,菜-菌轮作可降低真菌群落丰度,改变土壤微生物群落的结构组成,同时提高土壤中有益菌群的丰度,降低有害菌群的丰度。     

荔枝园间作平托花生对土壤理化性质、酶活性和细菌群落结构及多样性的影响

研究荔枝间作平托花生对荔枝园土壤理化性质、酶活性及细菌多样性的影响,为荔枝健康栽培提供理论依据和技术支撑。分别选取荔枝/平托花生间作和荔枝单作模式下10～20 cm土层的土壤样本,测定酸性有效磷、全磷和速效钾等理化因子的含量以及蔗糖酶、蛋白酶和脲酶等酶活性,并在此基础上,利用Illumina MiSeq测序平台对荔枝/平托花生间作模式和荔枝单作模式下土壤的细菌群落进行16S rRNA V4～V5区检测。结果表明:与荔枝单作模式相比,在理化因子方面,荔枝间作平托花生模式下的速效钾含量极显著提高138.9%,碱解氮含量显著降低19.6%,pH稍降低但无显著差异性;在土壤酶活性方面,蔗糖酶、酸性蛋白酶、脲酶、过氧化氢酶和多酚氧化酶均极显著或显著提高;在土壤细菌群落多样性方面,Chao1、ACE和Simpson指数分别显著提高5.5%、5.2%和3.7%。在土壤细菌群落结构方面,两组样本的主要优势菌门为酸杆菌门、变形菌门、绿弯菌门和放线菌门;主要优势菌属为Gp1、Gp2、Gp3、Gp4、Gp6、芽孢杆菌属和Gaiella等菌属;间作后变形菌门、放线菌门、红游动菌属、Gaiella和黄杆菌属相对丰度明显提高,酸杆菌门、芽孢杆菌属和Gp2相对丰度显著降低。冗余分析结果表明,碱解氮、pH、全磷和有效磷是影响土壤细菌菌属结构的主要理化因子;单作模式的土壤细菌群落结构差异主要是由pH的变化引起的,而间作模式细菌群落结构主要受碱解氮的影响;此外,在酸杆菌门亚群中,Gp1、Gp2、Gp3及Gp7与pH呈正相关;Gp4、Gp5、Gp6与pH呈负相关;芽孢杆菌属与碱解氮呈正相关;Gaiella、红游动菌属和黄杆菌属等绝大多数菌属均与理化因子呈负相关。因此,荔枝园间作平托花生不仅能改善土壤理化性质以及酶活性,还能提高土壤细菌丰富度和多样性,促进荔枝根系对营养元素的有效吸收,为农民有效利用荔枝园行间空地、实现荔枝健康栽培探索出一条新的路径。     

cry1Ac/cpti转基因大米水稻土中微生物群落的多样性研究

Two types of cry1Ac/cpti transgenic rice(GM1 and GM2)and their parental non-cry1Ac/cpti rice(CK1 and CK2)were planted in the field at Wufeng,Fujian Province,China for four years to investigate the influence of genetically modified rice on diversity of bacterial and fungal community in the paddy soil.The community composition and abundance of bacteria or fungi in the paddy soil were assessed at different growth stages of rice by denaturing gradient gel electrophoresis and real-time polymerase chain reaction based on 16S rRNA gene or SSU rRNA gene in the 4th year after the experimental establishment.The composition of bacterial or fungal community changed during rice growth,while no significant differences were observed between the fields cultivated with GM1and CK1,or between the fields cultivated with GM2 and CK2 in either bacterial or fungal community composition.The copy numbers of bacterial 16S rRNA gene in the soils with CK1,CK2,GM1 and GM2 ranged from 5.64×1011to 6.89×1011copies g-1dry soil at rice growth stages,and those of fungal SSU rRNA gene from 5.24×108to 8.68×108copies g-1dry soil.There were no marked differences in the copies of bacterial 16S rRNA gene or fungal SSU rRNA gene between CK1 and GM1 or between CK2 and GM2at any growth stage of rice.Planting cry1Ac/cpti transgenic rice had no significant effect on composition and abundance of bacterial and fungal community in paddy soil during the rice growing season at least in the short term.     

Application of DGGE analysis to the study of bacterial community structure in plant roots and in nonrhizosphere soil

To analyze the structure of bacterial communities in spinach roots and in the nonrhizosphere soil, we used PeR-amplified 16S rRNA gene fragments separated by denaturing gradient gel electrophoresis (DGGE). DGGE revealed a large number of band patterns, which were ascribed to various bacterial species composing each of the bacterial communities. The pattern from the roots was less complex than that from the soil. It is considered that DGGE analysis is suitable for studies of bacterial community structure in soil-plant ecosystems.     

沿海滩涂土壤中反硝化细菌群落随盐分梯度的更迭

To better understand the effect of salinity on denitrification communities,soils along a salinity gradient (ranging from 7.32 to 1.70 mS cm 1) in a wetland along the Yellow Sea coastline in Jiangsu Province,China,were studied using both culture-dependent and-independent methods.Culture efforts yielded 82 isolates in total,81.7% of which were close relatives of Bacillus sp.based on partial sequences of their 16S rRNA genes.Denaturing gradient gel electrophoresis (DGGE) analysis based on 16S rRNA sequences suggested possible existence of bacterial community succession along the salinity gradient.Clone library analysis based on nosZ gene sequences (coding nitrous oxide reductase) showed that operational taxonomic units (OTUs) associated with α-proteobacteria dominated in all three soils,whereas those associated with β-and γ-subdivisions showed a clear succession.In the high salinity soil,only the OTUs associated with α-subdivision were found.In the medium salinity soil,small proportions of β-(6.5%) and γ-associated (19.6%) OTUs were found.In the low salinity soil,the proportions were further increased to 33% and 25% for β-and γ-Proteobacteria,respectively.Statistic analysis using Unifrac P test showed that nosZ-communities in different saline soils were significantly different from each other.It could be concluded that α-subdivision of nosZ-community tended to be sustained in high salinity environments whereas β-and γ-subdivisions,especially the former,tended to be sustained in low salinity environments.Salinity was the key determinant of nosZ-community composition in the environment.     

Anammox反应器的启动及其菌群演变的研究

为了研究工艺条件对反应器内微生物多样性的影响,该论文采用城市污水处理厂活性污泥接种,通过培育硝化污泥,进行了启动厌氧氨氧化(Anammox)反应器的试验,并对启动过程中细菌的多样性变化作了跟踪研究。研究结果表明,以好氧活性污泥作为接种物,可成功地培育硝化生物膜;通过反应器运行条件的控制,硝化生物膜可从进行好氧氨氧化反应过渡到进行厌氧氨氧化反应。在此过程中,异养型细菌的数量大幅度下降,硝化细菌、反硝化细菌和厌氧氨氧化细菌的数量增大,推测它们都与厌氧氨氧化作用有关。运用PCR-DGGE技术证明,随着反应器运行时间的延长,菌群发生明显变化并呈现简化趋势。     

Effect of winter-flooding on methanogenic archaeal community structure in paddy field under organic farming

Paddy field is a major emission source of methane. Methane is the terminal product of anaerobic decomposition of organic matter and generated by methanogenic archaea under flooded conditions in paddy fields. This study aimed to reveal the effect of winter flooding on methanogenic archaeal community structure in paddy fields of Andosols under organic farming. Soil samples were collected from experimental paddy fields in the Field Science Center, Tohoku University, for two years. They were under flooding conditions during winter with organic farming, under non-flooding conditions during winter with organic farming and under non-flooding conditions during winter with conventional farming (non-organic farming). Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis of methanogenic archaeal 16S rRNA gene revealed that the DGGE patterns were nearly the same irrespective of the treatment and sampling times. Twenty-three bands were observed from each treatment and 4, 13 and 6 sequences were closely related to Methanomicrobiales, Methanosarcinales and Methanocellales, respectively. Real-time quantitative PCR analysis indicated that the abundance of methanogenic archaeal 16S rRNA gene and mcrA gene, encoding α subunit of methyl-coenzyme M reductase, was not significantly different among the paddy fields. This study first revealed a methanogenic archaeal community in an Andosol paddy field and showed that the community was not affected by winter flooding under organic farming.     

Relationships between the damage to radish caused by the root-lesion nematode Pratylenchus penetrans,its density prior to cultivation and the soil nematode community structure evaluated by polymerase chain reaction-denaturing gradient gel electrophoresis

Abstract

A preliminary survey using 20 conventionally farmed fields in which fumigants have been applied every year showed that the root-lesion nematode Pratylenchus penetrans was distributed both in the upper (0–30?cm) and lower (30–60?cm) soil layers. In six of the 20 fields, P.?penetrans was detected in the lower layers exclusively, suggesting that the most appropriate depth to sample soil is 0–60?cm to estimate the relationship between the density of P.?penetrans and its damage to radish. There was a highly significant correlation (r?=?0.923) between the density of P.?penetrans in the 0–60?cm depth and the number of spots on a radish. No damage to radish was observed in soils with <2.5 individuals of P.?penetrans per 20?g soil before cultivation. However, in cases in which the density of P.?penetrans was 3.4–6.2 individuals per 20?g soil, the number of spots on a radish showed more variation (0–131.5 per radish) and there was no significant correlation between them. The nematode community structure of soils with 3.4–8 individuals of P.?penetrans per 20?g soil, evaluated by polymerase chain reaction-denaturing gradient gel electrophoresis, was significantly different (anova, PC2, P?<?0.05) between soils with low (0–42) and high (more than 80) damage levels, suggesting that radish damage might be predicted on the basis of the prevailing soil nematode community structure.     

Relationships between the damage to radish caused by the root-lesion nematode Pratylenchus penetrans, its density prior to cultivation and the soil nematode community structure evaluated by polymerase chain reaction-denaturing gradient gel electrophoresis

A preliminary survey using 20 conventionally farmed fields in which fumigants have been applied every year showed that the root-lesion nematode Pratylenchus penetrans was distributed both in the upper (0–30 cm) and lower (30–60 cm) soil layers. In six of the 20 fields, P. penetrans was detected in the lower layers exclusively, suggesting that the most appropriate depth to sample soil is 0–60 cm to estimate the relationship between the density of P. penetrans and its damage to radish. There was a highly significant correlation ( r  = 0.923) between the density of P. penetrans in the 0–60 cm depth and the number of spots on a radish. No damage to radish was observed in soils with <2.5 individuals of P. penetrans per 20 g soil before cultivation. However, in cases in which the density of P. penetrans was 3.4–6.2 individuals per 20 g soil, the number of spots on a radish showed more variation (0–131.5 per radish) and there was no significant correlation between them. The nematode community structure of soils with 3.4–8 individuals of P. penetrans per 20 g soil, evaluated by polymerase chain reaction-denaturing gradient gel electrophoresis, was significantly different ( anova , PC2, P  <   0.05) between soils with low (0–42) and high (more than 80) damage levels, suggesting that radish damage might be predicted on the basis of the prevailing soil nematode community structure.     

Bacterial Communities Responsible for the Decomposition of Rice Straw Compost in a Japanese Rice Paddy Field Estimated by DGGE Analysis of Amplified 16S rDNA and 16S rRNA Fragments

To estimate the succession and phylogenetic composition of the bacterial communities responsible for the decomposition of rice straw compost under flooded conditions during the cultivation period of paddy rice, denaturing gradient gel electrophoresis (DGGE) analyses targeting 16S rDNA and 16S rRNA, followed by sequencing were conducted in a Japanese paddy field. The DGGE bands of the bacterial communities in the rice straw compost were significantly more numerous in the DNA samples than in the RNA samples. Although the band number of the DNA samples was almost constant throughout the period, RNA samples showed fewer DGGE bands after mid-season drainage than before it. Thus, about 81% of the bacteria present in rice straw compost were considered to be metabolically "active" before mid-season drainage and about 62% after it. The changes in the DGGE patterns of bacterial DNA and RNA before and after mid-season drainage, respectively, were also revealed by cluster analysis and principal component analysis of the DGGE patterns. These results indicated that the bacterial communities of rice straw compost incorporated into flooded paddy fields changed gradually along with the decomposition, except for the period of mid-season drainage, but that they were influenced by mid-season drainage. Members of β-, γ- and δ-Proteobacteria, Cytophaga-Flavobacterium-Bacteroides (CFB) group, Chlorobia, Verrucomicrobia, Chloroflexi, Spirochaetes, Firmicutes (clostridia) and Actinobacteria were present during the decomposition of rice straw compost. Characteristic "active" bacteria among them were as follows: Clostridium, Acinetobacter (γ-Proteobacteria) and β-Proteobacteria before mid-season drainage, Flavobacterium, Chondromyces , Chlorflexi and δ-Proteobacteria after mid-season drainage, and Spirochaeta and myxobacteria throughout the period.     

Abundance and community composition of methanotrophs in a Chinese paddy soil under long-term fertilization practices

Background, aim, and scope  As the second most important greenhouse gas, methane (CH₄) is produced from many sources such as paddy fields. Methane-oxidizing bacteria (methanotrophs) consume CH₄ in paddy soil and, therefore, reduce CH₄ emission to the atmosphere. In order to estimate the contribution of paddy fields as a source of CH₄, it is important to monitor the effects of fertilizer applications on the shifts of soil methanotrophs, which are targets in strategies to combat global climate change. In this study, real-time polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) based on 16S rRNA and pmoA genes, respectively, were used to analyze the soil methanotrophic abundance and community diversity under four fertilization treatments: urea (N), urea and potassium chloride (NK), urea, superphosphate, and potassium chloride (NPK), and urea, superphosphate, potassium chloride, and crop residues (NPK+C), compared to an untreated control (CON). The objective of this study was to examine whether soil methanotrophs responded to the long-term, different fertilizer regimes by using a combination of quantitative and qualitative molecular approaches. Materials and methods  Soil samples were collected from the Taoyuan Experimental Station of Agro-ecosystem Observation at Changde (28°55′ N, 111°26′ E), central Hunan Province of China, in July 2006. Soil DNAs were extracted from the samples, then the 16S rRNA genes were quantified by real-time PCR and the pmoA genes were amplified via general PCR followed by DGGE, cloning, sequencing, and phylogenetic analysis. The community diversity indices were assessed through the DGGE profile. Results  Except for NPK, other treatments of N, NK, and NPK+C showed significantly higher copy numbers of type I methanotrophs (7.0–9.6 × 10⁷) than CON (5.1 × 10⁷). The copy numbers of type II methanotrophs were significantly higher in NPK+C (2.8 × 10⁸) and NK (2.5 × 10⁸) treatments than in CON (1.4 × 10⁸). Moreover, the ratio of type II to type I methanotrophic copy numbers ranged from 1.88 to 3.32, indicating that the type II methanotrophs dominated in all treatments. Cluster analyses based on the DGGE profile showed that the methanotrophic community in NPK+C might respond more sensitively to the environmental variation. Phylogenetic analysis showed that 81% of the obtained pmoA sequences were classified as type I methanotrophs. Furthermore, the type I-affiliated sequences were related to Methylobacter, Methylomicrobium, Methylomonas, and some uncultured methanotrophic clones, and those type II-like sequences were affiliated with Methylocystis and Methylosinus genera. Discussion  There was an inhibitory effect on the methanotrophic abundance in the N and a stimulating effect in the NK and NPK+C treatments, respectively. During the rice-growing season, the type II methanotrophs might be more profited from such a coexistence of low O₂ and high CH₄ concentration environment than the type I methanotrophs. However, type I methanotrophs seemed to be more frequently detected. The relatively complex diversity pattern in the NPK+C treatment might result from the strong CH₄ production. Conclusions  Long-term fertilization regimes can both affect the abundance and the composition of the type I and type II methanotrophs. The inhibited effects on methanotrophic abundance were found in the N treatment, compared to the stimulated effects from the NK and NPK+C treatments. The fertilizers of nitrogen, potassium, and the crop residues could be important factors controlling the abundance and community composition of the methanotrophs in the paddy soil. Recommendations and perspectives  Methanotrophs are a fascinating group of microorganisms playing an important role in the biogeochemical carbon cycle and in the control of global climate change. However, it is still a challenge for the cultivation of the methanotrophs, although three isolates were obtained in the extreme environments very recently. Therefore, future studies will be undoubtedly conducted via molecular techniques just like the applications in this study.     

Community structure of methanogenic archaea in paddy field soil under double cropping (rice-wheat)

Community structure of methanogenic archaea in paddy field soil under double cropping (rice [Oryza sativa L.] and wheat [Triticum aestivum L.]) was studied by the denaturing gradient gel electrophoresis (DGGE) method. Soil samples under flooded and upland conditions were collected 7 and 6 times, respectively, from two paddy fields throughout a year, and two primer sets, 0357F-GC/0691R and newly designed 1106F-GC/1378R, were used for DGGE analysis. The 25 and 29 different bands were observed on the DGGE gels with the primers 0357F-GC/0691R and 1106F-GC/1378R, respectively. DGGE band patterns of the methanogenic archaeal community were stable throughout a year including the cultivation periods of rice under flooded conditions and of wheat under upland conditions. Cluster analysis and principal component analysis suggested that the difference in the soil type (sampling region) largely influenced the community structures of methanogenic archaea in paddy field soil, while the effects of sampling period and different fertilizer treatments on them were small. Most of the sequences obtained from the DGGE bands were closely related to Methanomicrobiales, Methanosarcinaceae, Methanosaetaceae and Rice cluster-I.