长期施肥对稻田土壤固碳功能菌群落结构和数量的影响

微生物固碳在减缓全球气候变化、实现人类可持续发展方面具有重要的意义,通过揭示长期不同施肥制度对土壤固碳细菌的影响规律,可以为我国稻田土壤科学施肥,稻田固碳和温室气体减排的共轭双赢作用提供重要的理论依据。以湖南宁乡国家级稻田肥力变化长期定位试验为平台,采用PCR-克隆测序和实时荧光定量(Real-time)PCR技术,研究不施肥(CK),氮磷钾肥（NPK）和秸秆还田（NPKS）3种长期施肥制度对稻田土壤固碳细菌群落结构及数量的影响。通过分析固碳细菌cbbL基因文库发现,长期施肥导致土壤固碳细菌种群结构产生了明显差异,NPK和NPKS处理中兼性自养固碳菌群落优势增加而严格自养固碳菌生长受到抑制。LUBSHUFF软件统计分析显示cbbL基因文库在CK、NPK及NPKS处理间均存在显著性差异。 3种施肥处理的稻田土壤细菌cbbL基因拷贝数为3.35?108 —5.61?108每克土,施肥后,土壤细菌cbbL基因数量增加,其中NPKS处理cbbL数量最多,是CK处理的1.5倍左右。稀疏曲线则显示长期施化肥导致细菌cbbL基因多样性高于NPKS,而NPKS高于CK。上述结果表明了长期施肥对土壤固碳细菌群落结构,多样性及数量均有显著的影响。本研究结果可为深入探讨稻田土壤微生物固碳潜力及其影响机理提供有力的依据。     

几种农田土壤中古菌、泉古菌和细菌的数量分布特征

真核生物、细菌和古菌三者共同构成了生命的三域系统.古菌作为第3种生命形式,不仅能在高温、强酸和高盐等极端环境下生存,而且在海洋、湖泊和土壤等生境中也广泛分布,预示其在整个生态系统中有着不可估量的作用.本文以2个农田剖面土壤和2个长期施肥试验站祁阳(QY)和封丘(FQ)的土壤为对象,以实时定量PCR方法为主要研究手段,对土壤中古菌(包括泉古菌)和细菌的16S rRNA基因拷贝数丰度变化进行了研究.结果表明:土壤泉古菌16S rRNA基因拷贝数要低于古菌l～2个数量级,两者与细菌相比,16S rRNA基因拷贝数大小顺序为土壤泉古菌＜古茵＜细菌,而古菌和泉古菌16S rRNA基因拷贝数与细菌的比值均随土壤深度加深而增大.不同施肥处理对土壤古菌和泉古茵的数量有显著影响.QY试验站土壤古菌和细菌的数量与土壤pH值显著相关(分别为r=0.850,P＜0.01和r=0.676,P＜0.05).FQ古菌、泉古菌和细菌与土壤pH值相关性不显著,与土壤有机质含量相关性均达显著水平(分别为r=0.783,P＜0.05;r=0.827,P＜0.05;r=0.767,P＜0.05).了解古菌包括泉古菌在农田土壤中的分布,可为评价其在生态系统和物质循环中的作用提供重要的理论依据.     

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.     

长期定位施肥对石灰性紫色水稻土古菌群落结构的影响

为了认识长期施肥对石灰性紫色水稻土培肥和肥力演化的作用,结合变性梯度凝胶电泳(denaturing gradient gel electrophoresis,DGGE)和限制性酶切片段长度多态性(RFLP)技术,研究了稻麦轮作下农家肥(M)、氮肥+农家肥(NM)、氮磷肥+农家肥(NPM)、氮磷钾肥+农家肥(NPKM)、无肥(CK)、氮肥(N)、氮磷肥(NP)、氮磷钾肥(NPK)等不同施肥制度对石灰性紫色水稻土古菌群落结构的影响.研究结果表明,长期定位施肥明显影响土壤中的古菌组成.在长期施用氮肥+农家肥、氮磷肥和氮磷钾肥+农家肥处理的土壤中,古菌多样性指数低于农家肥、氮磷肥+农家肥、无肥、氮肥和氮磷钾肥处理.在DGGE图谱的基础上,分别选择种植水稻和小麦的氮磷钾肥处理土壤样品,对古菌克隆子的16S rDNA序列进行了系统发育分析,发现水稻七古菌与各种土壤及水体环境的古菌极其相似.对DGGE图谱的聚类分析发现,不管是种植水稻还是小麦,8种施肥处理的古菌都聚在3个群里.种植水稻时,M和NPK处理下的土壤古菌聚成第一个群,NP处理下的聚成第二个群,另外5种施肥处理(NPKM,NM,Ck,N和NPM)聚成第三个群.种植小麦时,NPKM和M处理下的土壤古菌聚成一个群,NP处理下的聚成第二个群,N、NPK、NM、NPM和CK处理下的聚成第三个群.聚类分析结果显示,作物类型会影响土壤古菌群落结构.     

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。     

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。     

亚热带不同林分土壤氨氧化菌群落特征

为揭示亚热带不同森林类型对土壤氨氧化菌群落特征的影响,采用荧光定量PCR以及PCR-DGGE技术研究了阔叶林、杉木林、马尾松林和毛竹林土壤氨氧化古菌和细菌丰度及古菌群落结构特征.结果表明:不同林分土壤中氨氧化古菌数量(1.62×106~1.88×107个·g-1干土)高于相应土壤中的氨氧化细菌(2.41×105~4.36×105个·g-1干土);毛竹林土壤氨氧化古菌数量显著高于杉木林,而后者又显著高于阔叶林和马尾松林,但氨氧化细菌数量在不同林分之间没有显著差异.DGGE图谱分析表明,不同林分土壤中氨氧化古菌的物种有所差异,且毛竹林和杉木林土壤古菌群落结构迥异.氨氧化古菌在亚热带主要林分土壤中表现出明显优势,且除植被类型外,土壤速效钾、pH和有机质是引起土壤氨氧化古菌群落结构及多样性变异的主要因素.     

黄泥田土壤真菌群落结构和功能类群组成对施肥的响应

研究长期不同施肥黄泥田土壤真菌群落组成和功能特性,深入认识真菌对不同施肥的响应机理,可以为合理施肥和保证农田土壤健康发展提供科学依据.设置不施肥(CK)、单施无机肥(NPK)、无机肥配施农家肥(NPKM)、无机肥加秸秆还田(NPKS)4个处理,采用Illumina高通量测序和FUNGuild对不同施肥处理下黄泥田土壤真菌群落结构和功能群进行分析.结果表明:从门水平上看,土壤真菌群落主要由子囊菌、担子菌和接合菌构成,且以子囊菌为主(47%~74%).NPKM和NPKS处理中子囊菌的相对丰度分别为49%和47%,显著低于CK(71%)和NPK(74%)处理,从目水平上看,减少的主要为肉座菌、格孢腔菌和散囊菌.NPKM和NPKS处理担子菌相对丰度分别为18%和28%,高于CK(14%)和NPK(10%)处理,从目水平上看,增加的主要为银耳菌、糙孢伏革菌和伞菌.与CK相比,单施无机肥降低了担子菌含量.不同施肥处理均在一定程度上增加了接合菌门的丰度,从目水平看,以内囊霉菌和粪蛙霉菌为主.NPK处理真菌α多样性指数显著低于其他处理,NPKM和NPKS处理的丰富度指数(Chao1和ACE)高于CK和NPK处理.不同处理之间真菌的营养类型以腐生营养型为主(48%~57%),NPKM和NPKS处理的共生营养型真菌相对丰度为17%,显著高于CK和NPK处理,增加的以丛枝菌根真菌、外生菌根真菌为主.NPK处理动物病原菌含量(10%)显著高于其他处理.冗余分析(RDA)结果表明,土壤含水量、孔隙度和盐度是影响真菌群落结构和功能类群组成的主要因子,其次为有机质和总氮.长期单施无机肥降低真菌种群多样性,增加致病菌含量,不利于土壤健康,而有机无机肥配施可在一定程度上提高真菌种群丰富度指数和共生真菌比例,有利于保持黄泥田稻田土壤健康的生态环境和真菌群落的多样性.     

施肥模式对设施菜地根际土壤微生物群落结构和丰度的影响

以甘肃武威长期定位设施菜地为研究对象,设定CK(对照)、MNPK(有机肥和无机肥配施)、1/2MNPK(1/2有机肥和无机肥)、M(单有机肥)、NPK(单化肥)等5种施肥处理,采用末端限制性片段多态性分析(T-RFLP)和实时荧光定量PCR技术,研究了根际土壤中细菌、氨氧化细菌和nir K型反硝化细菌群落结构和丰度的变化。结果表明:施肥和根际效应均对设施菜地土壤中的细菌、氨氧化细菌和nir K型反硝化细菌群落结构和丰度产生影响。施肥模式影响根际细菌、氨氧化细菌和nir K型反硝化细菌的主要种群,但对不同功能微生物的影响有差异。细菌16S rRNA基因、amo A基因和nir K基因的最大丰度分别出现在M、MNPK和M处理,为每克干土6.60×109、1.85×108和3.49×107个拷贝数,是CK处理的2.74、2.81和3.46倍。土壤p H值、有机质和全磷含量是影响设施菜地根际细菌、氨氧化细菌和nir K型反硝化细菌丰度的关键因素。     

施氮对冬种紫云英不还田条件下稻田土壤微生物数量与结构的影响

揭示长期不同施肥制度对稻田土壤微生物的影响,为农田优化施肥和耕作制度提供理论依据。采用Illumina MiSeq高通量测序平台和荧光定量PCR技术,研究湖南省双季稻区连续8年不施肥(CK)以及冬种紫云英不还田条件下3种氮肥水平(N0、N100、N200)对紫云英盛花期土壤微生物群落数量和结构的影响。结果表明,与CK相比,冬种紫云英不还田配施氮肥显著降低土壤pH、硝态氮及土壤碳氮比(C/N),显著提高稻田土壤全氮、有机质和铵态氮含量。稻田土壤微生物数量在1.66×10~8～1.23×10~(10)拷贝数·g~(-1)干土,施肥均增加16S rDNA基因丰度,N0、N100、N200三种施肥水平处理的16S rDNA基因拷贝数分别是CK的1.0、74.4、32.6倍。3种氮肥水平处理的多样性指数(香农指数与辛普森指数)以及物种丰富度指数(Ace与Chao1)均高于CK,其中多样性指数最高的是N200,物种丰富度最高的是N100。不同样品16S rDNA的门水平分类的3个主要类群是变形菌门(Proteobac-teria)、绿弯菌门(Chloroflexi)和硝化螺旋菌门(Nitrospirae),分别占总OTU比例的33%～44.4%、17%～22.3%和9%～10%;N0、N100和N200土壤样品中变形菌门(Proteobacteria)和硝化螺旋菌门(Nitrospirae)的相对丰度均低于CK,而N0、N100和N200土壤样品中绿弯菌门(Chloroflexi)的相对丰度分别是CK的1.2、1.3和1.2倍。相关性分析表明,细菌16SrDNA基因拷贝数与土壤有机质、土壤总氮和铵态氮含量显著相关;土壤细菌菌群与多数土壤化学指标间存在密切的相关性。综上,冬种紫云英不还田施氮肥显著增加水稻产量和紫云英盛花期土壤微生物数量,改变土壤微生物菌群结构,改善了土壤化学性质,对湖南双季稻区科学合理种植绿肥具有指导意义。     

白蚁与动物及微生物的共生类型及其机制

综述了白蚁螱客的主要种类、共生关系及相关机制的研究进展。白蚁螱客中,已报道的动物种类达170种。在与动物的共生关系中存在偏利共生（宾主共栖和异种共栖）、互利共生和无关共生三种;在与微生物的共生关系中,存在与内生菌（原生动物、细菌、真菌和放线菌）和外生菌（蚁巢伞菌等）间的互利关系。指出了白蚁与螱客研究中存在的问题,给出了解决方案,并提出了今后可能的研究热点或方向,为白蚁的综合利用（如纤维素酶）及今后研究物种间的协同进化提供了基础资料。     

New amino-dithiaphospholanes and phosphoramidodithioites and their rhodium and iridium complexes

New sulfur derivatives of phosphoramidite ligands were synthesized and the impact of the sulfur unit on the spectroscopic properties of their rhodium and iridium complexes was investigated. The new ligands Bn₂NPSCH₂CH₂S^a(P-S^a) (Bn = benzyl, 4), Bn₂NPSCHCHS^a(CH₂)₃C^aH₂(P-S^a)(C^a-S^a) (6) and Bn₂NP(4-XC₆H₄OMe)₂ (X = S, 7a; X = O, 7b) were converted to the rhodium and iridium complexes trans-[Rh(CO)Cl(L)₂] (L = 4, 6, 7), [RhCl(COD)(L)] (L = 4, 6, 7), [IrCl(COD)(7a)] and [IrCl₂Cp∗(6)]. For comparison, some phosphoramidite complexes of these formulations also were synthesized. The new metal complexes were spectroscopically analyzed. For the carbonyl complexes, the ν_CO IR stretching frequencies were lower than for the corresponding phosphite and phosphoramidite ligands. The ¹J_PRh coupling constants for the rhodium complexes with the new ligands were also smaller than for the respective phosphoramidite and phosphite complexes. Finally, the ¹J_PSe coupling constants of the selenides of the new ligands were lower than those of the phosphoramidite ligands but higher than for PPh₃. The spectroscopic data reveal that the new thio ligands 4, 6 and 7a are more electron donating than phosphites and phosphoramidites but less electron donating than PPh₃.     

Astrocytes and Synaptosomes Transport and Metabolize Lactate and Acetate Differently

Astrocytes transport the monocarboxylate acetate, but synaptosomes do not. The reason for this is unknown, because both preparations express monocarboxylate transporters (MCT). The transport and metabolism of lactate, another monocarboxylate, was examined in these two preparations, and the results were compared to those for acetate. Lactate transport is more rapid in astrocytes than in synaptosomes, but of lower affinity (Kms of 17 and 4 mM, respectively). Lactate (0.2 mM) is metabolized to CO2 more rapidly in synaptosomes than in astrocytes (rates of 0.37 and 0.07 nmol x mg protein(-1) x min(-1), respectively). The reason for this is unclear, but cellular differences in lactate dehydrogenase isotype expression may be involved. Acetate is metabolized to CO2 more rapidly in astrocytes than in synaptosomes (rates of 0.43 and 0.02 nmol x mg protein(-1) x min(-1), respectively). This is likely due to cellular differences in the expression of monocarboxylate transporter subtypes.     

Rapporteur report: Basics and technology and metrology and standards

The first and second sessions of the Workshop focussed on the basics of ultrasound and infrasound, their applications in both industry and medicine, and metrology and protection standards for ultrasound applications.     

Cytoskeleton and mitochondrial morphology and function

It has been well established that the cytoskeleton is an essential modulator of cell morphology and motility, intracytoplasmic transport and mitosis, however cytoskeletal linkage to the organelles has not been unequivocally demonstrated. Indeed, cytoskeleton appears to be essential in determining and modulating gene phenotype as a function of cellular environment. According to recent studies, the organization of the cytoskeleton network together with associated protein(s) could be essential in regulating mitochondrial function and particularly the permeability of the mitochondrial outer membrane to ADP. The aim of this chapter is to summarize the main properties of the cytoskeletal environment of mitochondria and the possible role(s) of this network in mitochondrial function in myocytes.     

Relative and combined effects of ethanol and protein deficiency on strontium and barium bone content and fecal and urinary excretion

To elucidate accumulation of minerals in human iliac arteries with aging, the content of minerals was analyzed by inductively coupled plasma atomic emission spectrometry. Bilateral common, internal, and external iliac arteries of 16 men and 8 women, ranging ages from 65 to 93 yr, were examined. It was found that an extremely high accumulation of calcium and phosphorus occurred in the common iliac artery at old age, being higher than that of the internal and external iliac arteries. It should be noted that the accumulation of calcium and phosphorus is the highest in the common iliac artery among the human arteries examined to date. Regarding sexual differences, the content of calcium and phosphorus in the common and internal iliac arteries was higher in women than in men, whereas their content in the external iliac artery was lower in women than in men.     

Cross-reactivity between storage and dust mites and between mites and shrimp

Many patients have sensitivities to multiple species of storage and house dust mites. It is not clear if this is because patients have multiple sensitivities to species-specific mite allergens or if these mites share many cross-reacting allergens. Our objective was to further define the cross-allergenicity between several species of storage and house dust mites using crossed-immunoelectrophoresis (CIE), crossed-radioimmunoelectrophoresis (CRIE), immunoblotting, and ELISA. CIE and CRIE reactions revealed that storage mites shared two cross-antigenic molecules and one of these bound IgE in a serum pool from mite allergic patients. Antibody in anti-sera built to each species of mite recognized many SDS–PAGE resolved proteins of other mite species and this suggested the potential for other cross-reactive allergens. Among patient sera, IgE bound to many different proteins but few had IgE that bound to a protein with common molecular weights across the mite species and this suggested mostly species-specific allergens. Antiserum built to each mite species precipitated one protein in shrimp extracts that bound anti-Der p 10 (tropomyosin) and IgE in the serum pool. Anti-Der p 10 showed strong binding to shrimp tropomyosin but very little to any of the mite proteins. ELISA showed the mite extracts contained very little tropomyosin. The storage and dust mites investigated contain mostly species-specific allergens and very small amounts of the pan-allergen tropomyosin compared to shrimp and snail.     

Chemokines and cytokines: axis and allies in asthma and allergy

Allergic asthma can be precipitated by many factors. For the atopic person, fungus, pollen, dust mites, cockroach antigens, and diesel exhaust are all agents that may trigger an allergic attack. Cytokines and chemokines are integral mediators of fungal asthma. From the earliest time points, they recruit and activate the cells required for the clearance of fungus as well as being critical factors involved in the immunopathology of this disease. In the final analysis, it is clear that these mediators can act to the benefit or the detriment of the host.     

Oxidation and nitration of ribonuclease and lysozyme by peroxynitrite and myeloperoxidase

In spite of the many studies on protein modifications by reactive species, knowledge about the products resulting from the oxidation of protein-aromatic residues, including protein-derived radicals and their stable products, remains limited. Here, we compared the oxidative modifications promoted by peroxynitrite and myeloperoxidase/hydrogen peroxide/nitrite in two model proteins, ribonuclease (6Tyr) and lysozyme (3Tyr/6Trp). The formation of protein-derived radicals and products was higher at pH 5.4 and 7.4 for myeloperoxidase and peroxynitrite, respectively. The main product was 3-nitro-Tyr for both proteins and oxidants. Lysozyme rendered similar yields of nitro-Trp, particularly when oxidized by peroxynitrite. Hydroxylated and dimerized products of Trp and Tyr were also produced, but in lower yields. Localization of the main modified residues indicates that peroxynitrite decomposes to radicals within the proteins behaving less specifically than myeloperoxidase. Nitrogen dioxide is emphasized as an important protein modifier.