Production of a monoclonal antibody specific for a Flavobacterium species isolated from soil

Abstract Hybridomas secreting monoclonal antibodies (MABs) specific for a soil Flavobacterium species (P25) were isolated. The MAB (D10) was used to target P25 using an enzyme-linked immunosorbant assay (ELISA) and indirect immunofluorescence. Cross-reactivity of the MAB with other Gram-negative bacteria (including Flavobacterium spp.) and a number of Gram-positive bacteria was investigated but none were found. Cross-reactivity with other orange/yellow pigmented Gram-negative rods ( Pseudomonas/Flavobacterium type) isolated from the soil into which P25 has been introduced in field experiments was also assessed using a modified colony blotting procedure. None of the indigenous species tested were recognised by the monoclonal antibody, thereby allowing unambiguous identification of P25 in soil. The MAB D10 was shown to recognise P25 growth under low-nutrient or stored under starvation conditions, suggesting that the antigen is a constitutive component of the cell and that the microorganism should be detected in oligotrophic environments such as soil. The pattern of fluorescence of P25 gave a clear indication of the localisation of the antigen in the outer membrane/cell wall region, and this was confirmed by immunogold labelling. Preliminary studies on the limits of detection of P25 using immunofluorescence suggest that densities as low as 20 bacteria g⁻¹ soil can be enumerated.     

黄山松土壤可溶性有机质对氮添加的响应及其与细菌群落的关联

为探究黄山松土壤可溶性有机质(DOM)数量和质量对短期氮(N)添加的响应及其与细菌群落的关联,在福建戴云山自然保护区设置不同N添加水平(0、40和80 kg N·hm^-2·a^-1)试验,采用三维荧光与平行因子联用法,并结合高通量测序手段分别对土壤DOM和细菌群落进行分析。结果表明: 与对照相比,N添加整体降低了0～10和10～20 cm土层可溶性有机碳(DOC)含量和DOM腐殖化指数(HIX),其中,高氮(80 kg N·hm^-2·a^-1)添加下均显著降低。平行因子分析法进一步表明N添加下DOM中类腐殖质组分(C1、C2)的相对含量降低。此外,N添加减少了富营养细菌(变形菌门、酸微菌纲)的相对丰度,而增加了贫营养细菌(斯巴达杆菌纲)的相对丰度。富营养细菌的相对丰度与HIX、C1、C2呈显著正相关,与相对易分解的类富里酸组分(C3)呈显著负相关;而贫营养细菌的情况则相反。说明N添加下不同生活策略的细菌类群对DOM中难分解和易分解组分存在明显的偏好性。我们推测N沉降加剧背景下土壤微生物生活策略的转变可能有助于DOM组分的塑造。     

Seasonal fluctuations in the population of denitrifying and N2-fixing bacteria in an acid soil of a Norway spruce forest

The seasonal fluctuations in the concentration of cultured denitrifying and N₂-fixing bacteria were followed in an ammonium fertilised and a control soil of a Norway spruce forest near Villingen/Black Forest from December 1994 to August 1998. The horizontal distribution of bacteria in three layers was determined by the MPN-method and by molecular probing (colony hybridisation) using specific 0.4–0.7 kb DNA probes for denitrification steps (narG, nirS, nirK and nosZ) and for N₂-fixation (nifH). The data showed that highest bacterial counts and higher numbers of denitrifying and N₂-fixing bacteria were generally detectable in the upper (= 5 cm) soil layer and that their amount decreased with soil depth. The concentration of these cultured bacteria showed seasonal fluctuations with highest numbers in autumn/winter/early spring and with low counts in summer. Denitrifying and N₂-fixing bacteria amounted to less than 10% of the total number of cultured bacteria determined by the MPN-method. Fertilisation with ammonium did not cause a shift in the population of these bacteria. These findings were corroborated by hybridisation experiments with genomic DNA isolated from the different layers. Strongest DNA–DNA hybridisation band intensities were obtained in the upper soil layer and their intensities decreased with soil depth. Soil samples from Villingen assayed in the laboratory produced N₂O (in dependence of nitrate and C₂H₂ added to the vessels) and utilised this gas with higher activities in the assays with the fertilised soil. It is concluded that molecular techniques can successfully be applied for assessing seasonal fluctuations of bacterial populations in soil. Relative abundance of denitrifying and N₂-fixing bacteria can be determined from experiments with DNA isolated from soils. Attempts to transform these results to the total population of soil bacteria on a single cell basis are faced with many uncertainties.     

矿山复垦区土壤微生物及其生化特性研究

通过对矿区煤寿石凤化物上几种不同复垦措施的得垦区,进行一系列土壤微生物及其生化特性的分析研究,结果表明,各类和同生物菌群、数量以及生物活性强度、放线菌和真菌的优势种属、土壤养分等均 中植区大于不覆上区,豆科区大于禾本科区;而覆上厚度（１０～２０ｃｍ范围）对土壤养分、生理厚度（１０～２０ｃｍ范围）对土壤养分、生理群微生物及其活性等影响较小,可见覆江层土（１０ｃｍ左右）种植豆科植物是矿区复垦中一项快速     

浙江天台山不同林型土壤环境的微生物区系和细菌生理群的多样性

研究了天台山8种土壤环境的微生物区系,细菌生理群分布、组成和多样性。结果表明：黄山松林、竹林和云锦杜鹃林土壤中细菌、真菌和放线菌数量较多,而柳杉林土壤中较少。微生物数量与土壤有机质、全氮、全磷含量以及土壤凋落物的关系较大。每种土壤环境的细菌、真菌和放线菌占微生物总量的比例为：细菌数量最多,放线菌居中,真菌较少。土壤细菌生理群在天台山8种土壤环境中的分布有较大的差异。好气纤维素分解菌、好气固氮菌、氨化细菌、有机磷分解菌、无机磷分解菌在8种土壤环境中均占有较大的比例,是每种土壤环境的优势菌群,而反硝化细菌和反硫化细菌在每种土壤环境中占有的数量比例均相对较小,处于次要地位。七子花林、竹林、云锦杜鹃林和日本花柏林土壤细菌生理群的Simpson指数和Shannon-Wiener指数均较小,柳杉林、茶园、金钱松林、黄山松林土壤细菌生理群的Simpson指数和Shannon-Wiener指数相对较大。     

黄土丘陵区植被类型与土壤微生物区系及生物量的关系

以陕西延安黄土丘陵区5种不同植被类型(人工刺槐林、天然侧柏林、天然辽东栎林、灌丛和裸地)为研究对象,分析了土壤微生物生物量碳、氮含量、细菌和真菌的丰度变化规律及其与土壤基本化学性质的关系。结果表明:(1)4种植被类型的土壤质量较之裸地都有不同程度的改善,总体趋势:天然林地人工林地裸地;(2)土壤微生物生物量碳、氮的总体趋势:天然林地最高,人工林次之,裸地最低,且与土壤有机碳(SOC)、全氮(TN)和速效磷(AP)极显著正相关(P0.01);(3)裸地土壤的细菌丰度最低,人工刺槐林真菌含量显著低于天然辽东栎林。细菌丰度与土壤营养状况呈显著正相关(P0.05),而真菌与土壤营养无明显相关性,只与土壤pH负相关。说明在该研究区域,植被类型与土壤质量对微生物资源都具有不同程度的作用。     

黄河三角洲刺槐臭椿混交林与纯林土壤细菌群落结构和多样性特征分析

为研究黄河三角洲地区混交人工林土壤细菌群落特征,应用高通量测序技术,比较分析了刺槐臭椿混交林以及臭椿和刺槐纯林土壤细菌结构及多样性,并结合土壤理化性质进行分析。试验结果表明：在细菌门分类水平上,臭椿纯林、刺槐纯林、刺槐臭椿混交林土壤中分别检测出27、25、31门细菌,3种不同林分土壤中酸杆菌门、变形菌门、放线菌门、硝化螺旋菌门、绿弯菌门、浮霉菌门、芽单胞菌门、疣微菌门8种细菌是土壤中的主要细菌群落,其中酸杆菌门、变形菌门和放线菌门为优势细菌群落。不同类型人工林土壤中各门细菌相对丰度差异显著。混交林土壤细菌物种数和Shannon指数值分别为1910和9.1高于两种纯林。通过对土壤主要细菌群落与土壤理化性质进行主成分分析发现,3种不同林分之间在土壤细菌群落结构上有较高程度的分离,差异显著（P < 0.05）,有效磷含量与混交林土壤细菌群落有较强的正相关关系。因此可以得出结论,不同林分类型、土壤理化性质和细菌群落结构三者相互影响,刺槐臭椿混交增加了土壤细菌群落多样性,土壤理化性质在一定程度上影响土壤细菌结构和多样性。     

The ratio of fungi and bacteria in the biomass of different types of soil determined by selective inhibition

Tundra, chernozem (virgin and arable), soddy-podzolic (coniferous forest, meadow, and arable), and grey forest (larch forest) soils were used to separate the contributions of fungi and bacteria to substrate-induced respiration (SIR) with the help of antibiotics. For soils with a high content of organic matter (tundra and chernozem: 12 and 8%, respectively), the procedure of selective inhibition of SIR has been optimized. This procedure consists in application of high concentrations of streptomycin (50–120 mg/g of soil) and cycloheximide (50–80 mg/g of soil) and decreasing the weight of the analyzed soil sample. Soils under study have shown the predominant contribution of fungi (63–82%) to the total SIR. The fungal-bacterial ratio in the soils of natural ecosystems (0–5 cm, without litter) was 4.3, 2.2, 1.5, and 1.5 for tundra soil, virgin chernozem, coniferous (soddy-podzolic soil), and larch (grey forest soil) forests, respectively. The lower layers of soddy-podzolic (5–10 cm) and grey forest (48–58 cm) soils showed a decrease in the fungal and increase in the bacterial component in the total SIR.     

浙江天台山几种林型下土壤细菌生理群生态分布的特性

 对浙江省天台山8种土壤细菌生理群生态分布的特性进行了研究,结果表明：每种细菌生理类群在8种土壤中的分布有较大的差异,与土壤的立地条件、营养成分、水热条件有较大的关系。好气性纤维素分解菌、好气性固氮菌、氨化细菌、有机磷分解菌、无机磷分解菌在每种土壤中均占有较大的比例,为8种土壤细菌生理群的优势菌群。与云锦杜鹃(Rhododendron fortunei)林土壤相比,其余7种土壤细菌生理类群总体组成的相似顺序为：竹林土壤>日本花柏(Chamaecyparis pisifera)林土壤>金钱松(Pseudola     

桂北不同林龄桉树人工林土壤微生物数量和酶活性特征研究

为探讨桉树不同林龄土壤微生物活性的响应特征,该研究采用时空互代法,选择广西北部低山丘陵地区1～5年生桉树人工林为研究对象,分析了土壤微生物、酶活性在不同土层和季节的变化规律。结果表明:(1)土壤微生物和酶活性在土层中有明显的垂直分布特征,均随土层加深而趋于降低,且各土层间差异显著。(2)细菌、放线菌数量随季节的大小顺序为秋季>夏季>春季>冬季,真菌的变化规律为春季>夏季>秋季>冬季,而酶活性随季节变化表现为夏季、秋季活性较高,春季、冬季活性较低。(3)细菌、真菌、脲酶、过氧化氢酶随林龄增大表现出先减小后增大的趋势,放线菌则呈现先减小后增大再减小的趋势,而蔗糖酶、酸性磷酸酶随林龄的增大趋于增大。(4)林地土壤中三大类群微生物与四种土壤酶之间存在极显著正相关关系,说明土壤微生物与土壤酶活性相互影响,两者之间关系密切,共同影响土壤的质量。综上可知,不同土层土壤微生物和酶活性的季节响应特征差异较大,总体在冬季最低,主要与气温、水分条件、凋落物养分的归还等有关。不同季节、土层、林龄之间的交互作用对土壤微生物和酶活性有显著影响。     

Analysis of bacterial populations in a grassland soil according to rates of development on solid media

Abstract The process of colony formation by bacteria from grassland soil sampled in April, July and September was simulated by a colony-forming curve (CFC). The CFC was a super-imposition of several component curves (cCFC) given theoretically by the first order reaction (FOR) model [3,6]. The pattern of FOR model curves was not influenced by the time of sampling and four cCFCs were always recognized during an incubation period of 160 h. It was considered that the CFC describes an inherent property of the bacterial population of the field. Bacterial isolates were obtained from colonies produced in each of four cCFCs on agar plates. Isolates corresponding to one cCFC were classified as one group. The bacterial isolates were characterized by morphological and physiological tests and subsequently clustered. Few oligotrophic bacteria were obtained among bacteria which produced visible colonies within 63 h of incubation time. On the other hand, approx. 50% of bacteria which produced v colonies after 63 h were oligotrophic bacteria. The time required for the appearance of the first colony, t _r of the FOR model, was very similar in the isolates belonging to one group. A close linear relationship was observed between t _r value and doubling time of isolates.     

寡营养细菌及其在环境科学中的应用

寡营养细菌是生存在寡营养环境中的一类细菌,其多样性与生物量在整个生物圈组成中都具有较大的优势,因而在生物地球化学循环中发挥着重要作用.从20世纪80年代开始,寡营养细菌在自然或人为环境中的寡营养机制、对饥饿的生理反应以及在生态系统中的作用一直是微生物生态学研究的前沿领域之一,其理论价值与应用前景已受到各国微生物生态学家与环境科学家们的广泛重视.本文综述了寡营养细菌的基本概念、营养类型、生理生态特性、可能的寡营养机制、主要研究方法以及在医学细菌检测和环境重金属监测中的应用等,并指出了寡营养细菌在环境科学中的应用前景.     

A Most Probable Number method (MPN) for the estimation of cell numbers of heterotrophic nitrifying bacteria in soil

A Most Probable Number (MPN) method was developed allowing for the first time estimation of populations of bacteria capable of heterotrophic nitrification. The method was applied to an acidic soil of a coniferous forest exhibiting nitrate production. In this soil nitrate production was unlikely to be catalyzed by autotrophic nitrifiers, since autotrophic ammonia oxidizers never could be detected, and autotrophic nitrite oxidizers were usually not found in appreciable cell numbers. The developed MPN method is based on the demonstration of the presence/absence of nitrite/nitrate produced by heterotrophic nitrifying bacteria during growth in a complex medium (peptone-meat-extract softagar medium) containing low concentrations of agar (0.1%). Both the supply of the growing cultures in MPN test tubes with sufficient oxygen and the presence of low agar concentrations in the medium were found to be favourable for sustainable nitrite/nitrate production. The results demonstrate that in the acidic forest soil the microbial population capable of heterotrophic nitrifcation represents a significant part of the total aerobic heterotrophic population. By applying the developed MPN method, several bacterial strains of different genera not previously described to perform heterotrophic nitrification have been isolated from the soil and have been identified by bacterio-diagnostic tests.     

High genetic and physiological diversity of sulfate-reducing bacteria isolated from an oligotrophic lake sediment

The community structure of sulfate-reducing bacteria in littoral and profundal sediments of the oligotrophic Lake Stechlin (Germany) was investigated. A collection of 32 strains was isolated from the highest positive dilutions of most-probable-number series, and their partial 16S rRNA gene sequences and genomic fingerprints based on ERIC (enterobacterial repetitive intergenic consensus)-PCR were analyzed. The strains fell into eight distinct phylogenetic lineages, and the majority (70%) showed a close affiliation to the genus Desulfovibrio. Most of the remaining strains (22%) were related to the gram-positive Sporomusa and Desulfotomaculum groups. A high redundancy of 16S rRNA gene sequences was found within several of the phylogenetic lineages. This low phylogenetic diversity was most pronounced for the subset of strains isolated from oxic sediment layers. ERIC-PCR revealed that most of the strains with identical 16S rRNA gene sequences were genetically different. Since strains with identical 16S rRNA gene sequences but different genomic fingerprints also differed considerably with respect to their physiological capabilities, the high diversity detected in the present work is very likely of ecological relevance. Our results indicate that a high diversity of sulfate-reducing bacterial strains can be recovered from the natural environment using the established cultivation media. Received: 20 April 1998 / Accepted: 12 June 1998     

不同演替阶段岩溶石灰土可培养细菌的群落特征

岩溶土壤是岩溶生态系统的重要组成部分,其母质碳酸盐岩在气候、地形、时间及生物等因素的综合作用下可依次演替出黑色石灰土、棕色石灰土、黄色石灰土和红色石灰土。[目的] 了解可培养细菌群落对岩溶石灰土演替过程的响应,可为岩溶石漠化治理、生态恢复和重建等提供理论依据。[方法] 以黑色石灰土、棕色石灰土、黄色石灰土和红色石灰土为研究对象,对土壤SOC、TN和TP含量以及可培养细菌群落进行测定。[结果] 土壤SOC和TN含量表现为：黑色石灰土 > 棕色石灰土 > 黄色石灰土 > 红色石灰土,TP含量在黑色石灰土中最高,在红色石灰土中最低。本研究共分离纯化得到144株细菌,其中R2A培养基分离纯化出的细菌最多。细菌主要来自Actinobacteria、Bacteroidetes、Firmicutes和Proteobacteria门,Pseudomonas、Cupriavidus和Bacillus等是可培养菌中的优势属。在属水平上,黑色石灰土中分离得到的菌株最多并以Arenimonas、Thermomonas、Achromobacter、Brevibacillus等作为优势特有属。具有固碳、固氮和解磷功能的细菌主要分布在岩溶石灰土演替的早期。[结论] 基于母质碳酸盐岩特性,富含有机质的黑色石灰土在参与碳氮磷循环细菌的作用下形成。随着石灰土淋溶程度的增加,岩溶石灰土理化性质和可培养细菌多样性呈现降低的趋势。     

元阳梯田地方水稻品种根部内生菌及根际微生物的分离与鉴定

采用组织分离法和土壤稀释涂板法,对元阳哈尼梯田2个地方品种‘月亮谷’和‘红脚老粳’的根部内生细菌及根际土壤细菌进行了分离,研究元阳梯田传统水稻品种特殊的内生菌组成.结果表明: 试验共得到399个菌株.经形态特征及生理生化鉴定,月亮谷根部和其根际土壤分别分离到8和5个属,其中5个属是共有的;红脚老粳的根部和其根际土壤中分别分离到10和7个属,其中6个属是共有的.经分子生物学鉴定,月亮谷根部分离到11个种和5个属,根际土壤分离到8个种和4个属,其中5个种和4个属是共有的;红脚老粳根部分离到9个种和5个属,根际土壤分离到10个种和3个属,其中4个种和2个属是共有的.通过分子生物学鉴定,大部分菌株都可以鉴定到种,而通过形态及生理生化特性只能初步鉴定到属,但两种方法在属层次上的鉴定结果基本一致.元阳地方水稻根部内生细菌及根际土壤细菌具有一定的种属同源性与特异性.     

土霉素暴露对小麦根际抗生素抗性细菌及土壤酶活性的影响

通过培养的方法研究了土霉素暴露和小麦根际抗性细菌的数量、种类、分布特征及土壤酶活性之间的剂量效应关系。结果表明,土霉素暴露下小麦根际单一抗生素抗性细菌数量和抗土霉素—链霉素双重抗性细菌数都明显增加,且与暴露剂量呈正效应关系;同时,土壤磷酸酶、脱氢酶活性下降,但与土霉素的剂量效应关系不明显。从土霉素暴露的土壤中分离到50株抗性细菌,经形态观察、RFLP分组和16S rDNA序列测定与分析,将它们聚集在Actinobacteria、Bacilli、Alphaproteobacteria、Gammaproteobacteria 和Sphingobacteria类群。其中放线菌最多（15株）,占抗性菌总数的30 %;其次是Bacillus属细菌（9株）和Pseudomonas属细菌（8株）,分别占18 %和16 %。同时,具有抗性的人类机会致病菌Pseudomonas、Sphingomonas和Stenotrophomonas属细菌在土霉素暴露的样品中均被分离到,分别占抗性菌株总数的16 %、8 %和4 %。值得注意的是,随着土霉素暴露剂量的增加,小麦根际优势促生菌Bacillus属细菌的抗性检出率逐步降低;但具有抗生素抗性的人类机会致病菌Pseudomonas、Sphingomonas和Stenotrophomonas属细菌的检出率却明显增加,提示可能会进一步增大其机会致病性。     

Morphometric analysis of bacteria associated with soil millipedes

Scanning electron microscopy revealed that the average cell size of bacteria associated with the digestive tract of soil millipedes was 0.65 μm in diameter, 1.36 μm in length, and 0.60 μm³ in volume. An example of millipedes illustrated that the intestinal tract bacteria of soil invertebrates share the following features: (1) a high density level in this habitat; (2) existence mostly in the form of vegetative cells; (3) a cell size significantly smaller than that of bacteria functioning in soil; (4) a cell size closer to the lower limits of the size range characteristic for bacterial cultures grown in laboratory media. All this suggests that the bacterial community of the digestive tract differs from the typical soil community not only in composition but also in a higher level of physiological activity.     

从两类不同培养基上分离的土壤细菌的特征比较

通过对两类不同培养基上分离的土壤细菌的生理特征的比较,发现两组细菌的种群有所不同.芽孢杆菌主要生长在宫营养的培养基上,而很难在低营养的培养基上生长.假单孢杆菌和棒杆类型的细菌则在两类培养基上均可生长.这与两类细菌对环境的适应能力有关.     

Population study of atmospheric bacteria at the Fengtai district of Beijing on two representative days

Summary We collected bacteria from the atmosphere (5–6 m above ground level) with an Andersen microbial sampler at Fengtai District, Beijing, in the spring and autumn of 1989. The 195 strains of bacteria collected and isolated were identified according to their morphological, biochemical and physiological characteristics. The results showed that there were 11 bacterial genera in the atmosphere of the town, the most abundant beingStaphylococcus andBacillus. The order of prevailing genera of the two seasons is similar. In morphology, gram positive (G⁺) bacteria predominate among atmospheric bacteria (G⁺ cocci, G⁺ rods and G⁺ endospore-forming rods). It seems that the constitution of atmospheric bacteria is influenced not only by the source strength of the various bacteria in the environment, but also by their somewhat resistant mechanisms such as protective pigments and spore structure. From our experience, in order to avoid the possible data loss from selecting and identifying some «representative» colonies of samples, all strains isolated should be identified when studying ecological distribution of microbes in the atmosphere.