luxAB基因标记甲基对硫磷降解菌DLL—1在土壤和植株根部的生态行为研究

发光酶标记是1种有效的跟踪微生物在生态环境中动态行为的技术手段。采用luxAB基因标记技术对甲基对硫磷降解菌DLL-1在土壤中的分布和植株内的定殖情况进行了研究。结果表明，DLL-1可以较长时间的在植株根际定殖，30d后仍可用X-感光片检测到菌体的存在，而根际外未检测到DLL-1。植株根剖开后在培养基上培养24h后进行X-光片曝光，发现DLL-1能够进入植株内并定殖。菌株回收后采用测定其农药降解活力和检查质粒图谱2种方法证实，所观测的回收菌株就是接种的DLL-1菌株。     

甲基对硫磷降解菌DLL-1的发光酶基因标记及在土壤中的变化

采用三亲接合法成功地将携带有发光酶基因的pTR1 0 2转入了甲基对硫磷降解菌Pseudomonassp .DLL -1菌株中。获得的接合子在液体培养、固体培养过程中以及在土壤中其标记质粒非常稳定。标记质粒并没有给受体细胞的生长、降解甲基对硫磷的能力带来严重的影响。因此 ,将标记菌株DLL -1-B用于土壤生态研究是可行的。在土壤中 ,DLL -1 -B的数量在接种后最初 2天下降幅度较小 ,随后下降速度较快 ,但当下降到 1 0 3～ 1 0 4 g- 1时趋于稳定。DLL -1 -B在水中下降速度更快 ,存活时间更短。DLL -1 -B在旱地土壤中的移动性较差 ,大多存在于 0～ 1 0cm土壤中。在水田中 ,DLL -1 -B的移动性较好 ,2周后 ,2 0～ 3 0cm土壤中测得DLL -1 -B为 2× 1 0 2 g- 1。     

3株芽孢杆菌在水稻根际定殖促生及其在土壤中的存活

植物根际促生菌(PGPR)可分泌植物生长激素,促进土壤养分循环,是生物肥料重要的种质资源。PGPR吸附于作物根系是形成定殖与功能的必要条件,抗性药物标记是研究功能菌株定殖促生能力的一种手段。利用解钾功能菌(Bacillus aryabhattai LZP01,简称LZP01)、解磷功能菌(Bacillus pumilus LZP02,简称LZP02)和溶磷功能菌(Bacillus megaterium LZP03,简称LZP03)这3株高效的水稻根际促生菌,通过抗生素药物利福平梯度筛选分别得到标记菌株K-LZP01、K-LZP02和K-LZP03,并且研究标记菌株的功能保留情况,进而检测标记菌株在水稻根际与土壤中定殖存活及促生能力。结果表明,水稻幼根浸入菌液后吸附量随浸入时间的增加而增大,其中K-LZP01在10min后吸附基本稳定;K-LZP01在50 min时吸附量达到最大;K-LZP03在5 min时吸附量达到最大,最大吸附量为12×10~7CFU·g~(-1),之后趋于动态稳定。将水稻幼苗浸入不同浓度菌悬液中,幼根吸附量随菌悬液浓度的增大而增大。标记菌株在接种于土壤中10 d后定殖量趋于稳定,且定殖能力较强。距离水稻根际越近标记菌株含量越高,表明菌株定殖竞争力越强。在灭菌土与未灭菌土条件下,3株菌株均具有促进水稻根系生长的能力。     

外源降解菌对黄麻根区净化能力的生物强化作用

通过原生质体电融合得到经EGFP标记的外源细菌(蒽高效降解菌An和表面活性剂产生菌P)与黄麻根际优势菌融合的2株融合子Tu-An与融合子Tu-P,然后将两种融合子以及出发菌株分别定殖到播种了黄麻(Corchoruscapsulari)的蒽污染土壤中,对不同污染物浓度下的细菌定殖、植物生长及蒽降解做了初步研究.研究表明,在相同的初始接种量下,融合子的定殖数量明显高于出发菌株;在两种融合子共存的条件下,定殖数量也略高于单一融合子的定殖数量.在接种外源菌的土壤中,植物获得了更好的生长条件,得以良好生长;而没有投加降解菌的组别中,黄麻的生长明显受到了较严重的抑制.投加外源降解菌可以促进生物降解,达到生物强化的目的.实验研究说明,把外源降解菌投加到污染土壤中进行生物强化修复是可行的.图3表2参20     

玉米内生固氮菌的回接分离及限菌条件下在玉米根内的定殖

对从玉米茎内分离的18株固氮菌进行了回接分离试验，经多碳源无氮培养基、菌落形态、颜色与REP－PCR相结合的方法，筛选、鉴定出了两株具有较高乙炔还原活性和能在玉米体内定殖的菌株R1和R6。把携带gfp标记基因的质粒pKK223-GFP和nifH-gfp的质粒pMGFP2.1分别转化R1和R6，得到携带gfp，且菌落带绿色荧光的转化子R1A和R6A，以及携带nifH-gfp的转化子R1B和R6B。在限菌条件下对R1A和R6A，在玉米根内的定殖以及R1B和R6B中nifH-gfp融合基因的表达进行了研究，结果表明了R1A和R6A主要定殖在根的皮层细胞、胞间隙、中柱细胞和导管内，有时在活的细胞内也可检测到，R1B和R6B中的融合基因因受玉米根内营养以及其它与固氮酶基因表达相关因素的限制，很难在玉米根内表达，只有在添加了1‰蔗糖后，nifH-gfp才能在根内表达。     

DLL-1菌对甲基对硫磷农药的降解作用及其降解机理

研究了假单胞菌 (DLL - 1)在水溶液介质中降解甲基对硫磷的性能与影响因素及其降解机理。结果表明 ,当菌体浓度为 10 5个·ml-1时 ,即发生快速的降解作用 ,3h时 ,降解率达 88.5 %。在pH为 5 .0、7.0和 9.0条件下 ,DLL - 1菌均产生对甲基对硫磷农药的高效降解作用。采用气相色谱 -质谱联用技术与离子色谱法 ,测定了DLL - 1菌 -甲基对硫磷作用过程产生的中间产物和最终产物 ,表明对硝基苯酚为主要中间产物 ,且DLL - 1菌能将其进一步降解为NO2 -和NO3 -。     

绿色荧光蛋白基因标记内生枯草芽孢杆菌

用枯草芽孢杆菌168菌株rpsD基因的启动子替换质粒pGFP4412中蜡状芽孢杆菌4412启动子,从而构建了能在枯草芽孢杆菌中表达绿色荧光蛋白基因gfpmut3a的载体pS4GFP,将其导入具有内生、防病、促生作用的野生型枯草芽孢杆菌BS-2菌株中,筛选获得遗传稳定性好且具有良好发光表型的标记菌株BS-2-gfp.该标记菌株在小白菜体内的定殖研究结果表明,该菌株能够在小白菜根际及根、茎、叶内定殖和传导,接菌50d后仍能在其体内分离到标记菌株.图5参17     

龙葵根际和内生Cd抗性细菌的筛选及其生物学特性

调查了南京栖霞铅锌银矿区龙葵根际土壤和植株的重金属含量,采用稀释培养法和选择性培养基分离筛选Cd抗性细菌并研究其生物学特性.结果表明,栖霞山铅锌银矿区龙葵根际土壤总Cd含量平均为19.74 mg·kg -1,污染十分严重,龙葵(Solanum nigrum)茎和叶的Cd富集系数分别为1.21 ～9.65和0.65 ～4.76,对Cd具有较强富集能力.从龙葵根际土壤和植株中分离筛选到5株Cd抗性细菌,菌株AR1和AY1分别分离自根和叶内,BGJ4和CGJ1均分离自根际土.16S rDNA序列分析表明5株Cd抗性细菌均属于芽孢杆菌属(Bacillus),能够产生吲哚乙酸(IAA)和铁载体,根内生细菌AR1在含Cd2+ 30 mg·L-1的培养基中仍能生长良好.在1.5 mg· L-1Cd2+污染条件下,Cd抗性细菌AR1、AY1、BGJ4菌株能够明显促进油菜(Oilseed rape)幼苗根的伸长,在植物-微生物联合修复重金属污染土壤方面具有潜在的应用前景.     

根际微生物耦合降解系统的构建及其对蒽污染土壤的修复

摘要将增强型绿色荧光蛋白(EGFP)标记的黄麻土生根际优势菌Tu-1B分别与葸高效降解菌An-2和生物表面活性剂产生菌P7-50进行原生质体电融合，得到两株具有荧光标记的融合子Tu-An和Tu-P．将二融合子接种于植物根际土壤，构建根际微生物耦合降解系统，：在40d时该系统的最大降解率为96％．对根际土壤中的Tu-An融合子进行了检测，结果表明融合子在根际能稳定旺盛生长．图3表4参18     

不同纤维素降解菌对玉米秸秆的降解效果

该研究旨在探索不同纤维素降解菌对玉米秸秆降解效果的差异性,为秸秆资源化利用提供参考。利用筛选自宁夏秸秆堆、畜粪和土壤中的四株纤维素降解菌,分别为黄曲霉(Aspergillusflavus)Z5-3菌株,非洲哈茨木霉(Trichoderma afroharzianum)Z6-4菌株,藤黄单胞菌(Luteimonas sp.)X11-1菌株,施氏假单胞菌(Pseudomonas stutzeri)X3-5-1菌株,通过室内控温秸秆堆肥发酵试验,研究添加不同纤维素降解菌后堆肥温度、pH值、碳损失、氮损失、有效养分含量的变化规律。试验结果表明,与对照(不加菌处理)比较,加菌处理提前1-2 d进入高温期,最高温度提高了0.3-1.3℃,且细菌处理较真菌处理提前1 d进入高温期,最高温度提高了0.5-0.7℃。pH值总体呈先增加后降低的趋势,且在高温期达到最大值。与对照相比,加菌处理NH3和CO2累积释放量具有降低趋势。以全氮含量变化计,细菌X11-1、X3-5-1处理的氮损失分别为26.14%和26.57%,真菌Z5-3、Z6-4处理分别为36.49%和34.19%,表明添加不同纤维素降解菌可不同程度地降低堆肥物料中的碳损失和氮损失,其中细菌处理保氮效果显著优于真菌处理。与对照相比,细菌X11-1、X3-5-1处理、真菌Z5-3、Z6-4处理分别提前腐熟5、2、2、2d,表明添加纤维素降解菌可减少堆肥物腐熟时间,促进物料养分浓缩。综合判断,玉米秸秆堆肥中加入纤维素降解菌,有利于降低物料的碳、氮损失,且细菌的保氮效果优于真菌,其藤黄单胞菌X11-1处理最先达到腐熟,为最佳处理。     

Effects of anaerobiosis on root metabolism of Zostera marina (eelgrass): implications for survival in reducing sediments

The temperate seagrass Zostera marina L. typically grows in highly reducing sediments. Photosynthesis-mediated O₂ supplied to below-ground tissues sustains aerobic respiration during photosynthetic periods. Roots, however, experience daily periods of anoxia and/or hypoxia at night and under conditions that reduce photosynthesis. Rhizosphere cores of Z. marina were collected in August 1984 from Great Harbor, Massachusetts, USA. We examined short-term anaerobic metabolism of [U-¹⁴C]sucrose in excised roots and roots of intact plants. Under anaerobic conditions roots showed appreciable labeling of CO₂, ethanol and lactate, and slight labeling of alanine and other metabolites. Over 95% of the ¹⁴C-ethanol was recovered in the root exudate. Release of other metabolites from the roots was minimal. Ethanol was also released from hypoxic/anoxic roots of intact plants and none of this ethanol was transported to the shoot under any experimental conditions. Loss of ethanol from roots prevented tissue levels of this phytotoxin from increasing during anaerobiosis despite increased synthesis of ethanol. Anaerobic metabolism of [U-¹⁴C]glutamate in excised roots led to appreciable labelling of -aminobutyrate, which was known to accumulate in eelgrass roots. Roots recovered to fully aerobic metabolism within 4 h after re-establishment of aerobic conditions. The contributions of these root metabolic responses to the ability of Z. marina to grow in reducing marine sediments are related to light-regulated interactions of shoots and roots that likely dictate depth penetration, distribution and ecological success of eelgrass.     

A simulation model to evaluate the impacts of invasive earthworms on soil carbon dynamics

Recent studies have reported that earthworm invasions alter native communities and impact nutrient cycling in terrestrial ecosystems. We developed a simulation model to evaluate the potential impacts of earthworm invasions on carbon dynamics, taking into consideration earthworm feeding strategies and priming effects on the microorganisms through their casting activities. Responses of carbon stocks (forest litter, soil organic matter, microbial biomass and earthworm populations) and carbon fluxes (litter decomposition, earthworm consumption, and microbial respiration) were used to evaluate an earthworm invasion of a forest ecosystem. Data from a northern temperate forest (Arnot Forest, New York) were adapted for model calibration and evaluation. Simulation results suggest that the impact and outcome of earthworm invasions are affected by pre-invasion resource availability (litter and soil organic matter), invasive earthworm assemblages (particularly feeding strategy), and invasion history (associated with earthworm population dynamics). The abovementioned factors may also determine invasion progress of earthworm species. The accuracy of the model could be improved by the addition of environmental modules (e.g., soil water regimes), precise parameters accounting for individual species attributes under different environmental conditions (e.g. utilization ability of different types of food resources), as well as earthworm population dynamics (size and structure) and interactions with predators and other invasive/indigenous species during the invasion progress. Such an earthworm invasion model could provide valuable evaluation of the complicated responses of carbon dynamics to earthworm invasions in a range of forest ecosystems, particularly under global change scenarios.     

发根农杆菌T—DNA基因对3种葛属植物毛状根形态和葛根素含量的影响

野葛、山葛和三裂叶葛的离体叶片与发展根农杆菌R1601直接感染诱导出毛状根并能够合成葛根素，毛状根离体培养后均出现两种形态：一种是有较高的葛根素含量的典型毛状根，另一种是葛根素含量低但生长速度较快的愈伤组织化毛状根，两类毛状根均有rol/B基因存在，表明rolB基因对所有毛状根的诱导和生长是必需的，aux1存在于所有愈伤组织化毛状根，而在典型毛状根中的检出率为20％－50％，表明aux1基因有诱导毛状根产生愈作组织的作用，含有ags基因的毛状根葛根素含量低于不含ags基因的毛状葛根素含量，表明ags基因不利于次生代谢合成，表3参12     

入侵植物南美蟛蜞菊营养器官的形态解剖研究

南美蟛蜞菊（Wedelia trilobata（L.）Hitchc.）是华南地区常见的入侵植物之一,其环境适应性强,繁殖速度快,对入侵地生态环境的破坏而导致生物多样性丧失的危害现象已经引起关注。结构与功能的相适应是生物学的基本观点之一,对入侵植物营养器官的内部形态解剖结构的研究,是理解入侵能力与其生物特性之间关系的直接路径,同时也是其他生理机理研究的结构基础。为研究入侵植物南美蟛蜞菊营养器官内部形态结构与其入侵能力的适应性,采用常规徒手切片技术对其根、茎及成熟叶片3大营养器官进行解剖及显微观察。结果表明：南美蟛蜞菊根、茎均具有次生结构。根韧皮部外方薄壁细胞具有分泌道,次生结构横切面中央为发达的次生木质部所填充,周皮代替表皮起保护作用,根的初生生长时期长,具有次生生长可视为其入侵定居时与本地物种形成地下资源和空间竞争的结构基础;茎的初生结构分化不久即产生次生结构,次生结构中央有明显的髓,次生维管束组织产生于初生结构的维管束之间并形成一管状结构明显将皮层和髓分开,茎内部组织高度木质化可视为是其茎直立生长及竞争地上资源和空间的结构基础;叶片为异面叶,上下表皮均具有气孔器和表皮毛,叶片内部具有分泌道,维管束发达且具有束鞘延伸,能与叶片表皮细胞共同构成辅助输导系统,叶片的结构特征是构成其喜阳植物的基础。此外叶片及根分泌道的存在可能与其化感物质的分泌有关。研究结果丰富了南美蟛蜞菊入侵适应性研究的背景基础,同时也弥补其在形态结构研究中的空缺。     

The invasive grass Agropyron cristatum doubles belowground productivity but not soil carbon

Root dynamics are among the largest knowledge gaps in determining how terrestrial carbon (C) cycles will respond to environmental change. Increases in productivity accompanying plant invasions and introductions could increase ecosystem C storage, but belowground changes are unknown, even though roots may account for 50-90% of production in temperate ecosystems. We examined whether the introduction of a widespread invasive grass with relatively high shoot production also increased belowground productivity and soil C storage, using a multiyear rhizotron study in 50-year-old stands dominated either by the invasive C3 grass Agropyron cristatum or by largely C4 native grasses. Relative to native vegetation, stands dominated by the invader had doubled root productivity. Soil carbon isotope values showed that the invader had made detectable contributions to soil C. Soil C content, however, was not significantly different between invader-dominated stands (0.42 mg C/g soil) and native vegetation (0.45 mg C/g soil). The discrepancy between enhanced production and lack of soil C changes was attributable to differences in root traits between invader-dominated stands and native vegetation. Relative to native vegetation, roots beneath the invader had 59% more young white tissue, with 80% higher mortality and 19% lower C:N ratios (all P < 0.05). Such patterns have previously been reported for aboveground tissues of invaders, and we show that they are also found belowground. If these root traits occur in other invasive species, then the global phenomenon of increased productivity following biological invasion may not increase soil C storage.     

西葫芦对土壤持久性有机氯化物污染的指示性研究

西葫芦(Cucurbita pepo L.)对土壤中持久性有机氯化物的超强吸收能力已被证实,意味其具有指示区域土壤持久性有机氯化物污染状况的潜能。本研究采用农田小区试验,考察了西葫芦不同组织器官(根、过渡茎、茎、叶和果实)在6个生长期对有机氯化物的累积吸收行为;采用同位素稀释高分辨气相色谱-高分辨质谱法,分析了种植土壤及西葫芦各组织器官中的21种有机氯农药(OCPs)和18种多氯联苯(PCBs)。结果表明,西葫芦可将根吸收的OCPs和PCBs传递到过渡茎和茎。不同生长期采集的西葫芦根、过渡茎和茎中的OCPs和PCBs浓度基本稳定,无明显生物稀释效应,且此3个组织器官中OCPs和PCBs的分布模式与土壤中的分布模式基本一致。因此,可以用西葫芦根和茎中持久性有机氯化物的浓度指示土壤中持久性有机氯化物的污染水平,根和茎的采样时间可以不受西葫芦生长期的限制。     

Nitrogen turnover in the leaf litter and fine roots of sugar maple

In order to better understand the nitrogen (N) cycle, a pulse of 15NO3- was applied in 1998 to a sugar maple (Acer saccharum) dominated northern hardwood forest receiving long-term (1994-2008) simulated atmospheric N deposition. Sugar maple leaf litter and live fine-root 15N were quantified for four years prior to labeling and for 11 subsequent years. Continuous sampling of 15N following addition of the tracer enabled calculation of leaf litter and fine-root N pool turnover utilizing an exponential decay function. Fine-root 15N recovery peaked at 3.7% +/- 1.7% the year the tracer was applied, while leaf litter 15N recovery peaked in the two years following tracer application at approximately 8%. These results suggest shoots are primarily constructed from N taken up in previous years, while fine roots are constructed from new N. The residence time of N was 6.5 years in leaf litter and 3.1 years in fine roots. The longer residence time and higher recovery rate are evidence that leaves were a stronger sink for labeled N than fine roots, but the relatively short residence time of tracer N in both pools suggests that there is not tight intra-ecosystem cycling of N in this mature forest.     

Dynamics of the epibenthic community on roots of the mangrove Rhizophora mangle,at Bahía de Buche,Venezuela

The community of epibenthic organisms growing on the roots of the red mangrove Rizophora mangle was studied from March 1976 to September 1977, at a site 100 km east of Caracas, Venezuela. Recruitment was relatively constant and low in intensity for most species. Asbestos panels submerged in March–April 1976 and November 1976 were similar in species composition by September 1977; species composition of panels submerged in March–April 1976 did not change greatly between November 1976 and September 1977. There was little change in the mean species composition on ca. 120 roots during the 18 month study period. The major feature of the community was the large differences in species composition among roots. Even species which were rare in the community as a whole were common on some roots. I ascribe these differences to chance resulting from the low recruitment rate of most species and the low rate of supply of new roots (8% yr^-1). Once present on a given root, species seem to live for many years and resist the invasion of most other larvae. The initial differences in species composition among roots persists and the overall community does not change greatly with time. Predation had little effect on community development.     

利用GFP和抗性双类型标记监测联合固氮菌在玉米根际的定殖

将来自质粒ｐＫＲＰ１０、ｐＫＲＰ１１和ｐＫＲＰ１２的氯霉素、卡那霉素和四环素抗性基因分别插入质粒ＧＦＰｍｕｔ２中ｇｆｐ基因下游的ＰｓｔＩ位点,得到ｇｆｐ和不同抗性基因共存的重组质粒,转化Ｅｎｔｅｒｏｂａｃｔｅｒ ｇｅｒｇｏｖｉａｅ ５７－７野生型菌株和耐铵工程菌Ｅ７后,得到既有抗生素抗性又在蓝光下呈现亮绿荧光的菌株。用它们接种玉米后,利用这两种选择标记双重筛选重新分离到的细菌确定了接种菌在玉米幼苗