Soil DNA extraction and assessment of the fate of Mycobacterium chlorophenolicum strain PCP-1 in different soils by 16S ribosomal RNA gene sequence based most-probable-number PCR and immunofluorescence

Since Mycobacterium chlorophenolicum strain PCP-1 is not detectable in soil by selective plating, a specific tracking method was based on the polymerase chain reaction (PCR) using soil DNA as a target. A direct extraction protocol based on bead beating was adapted and used to obtain PCR-amplifiable DNA from five different soils. In one soil, the disruption of cells of PCP-1, of Pseudomonas fluorescens R2f and of Paenibacillus azotofixans P3L5, as well as of the indigenous bacteria increased with increasing bead beating times. After 4.5 min, lysis efficiency was about 90% or more in all cases. Total DNA yields varied between soils, from 2 to 35 μg g^–1. The purification steps needed to obtain amplifiable DNA were different per soil. To detect target DNA specifically in bacterial cells, a new indirect extraction protocol was developed, which efficiently dislodged bacterial cells from the soil matrix, and produced amplifiable DNA with high yield. To detect strain PCP-1 in soil, 16S ribosomal gene-based PCR combined with oligonucleotide hybridization was applied using a most-probable-number (MPN) set-up, whereas immunofluorescence was used for calibration. Strain PCP-1 was detected shortly after introduction into three soils at about the inoculum levels, as evidenced by both approaches. Both the direct and indirect DNA extraction methods yielded similar MPN estimates. The dynamics of M. chlorophenolicum PCP-1 was estimated in two soils over 14 days via MPN-PCR/oligonucleotide probing. PCP-1 showed good survival in both soils, and results obtained by MPN-PCR with directly and indirectly extracted DNA were internally consistent. Immunofluorescence cell enumerations supported the gross stability of PCP-1 in these two as well as in two additional soils. Received: 8 February 1996     

A rapid diagnostic assay for detection and quantification of the causal agent of strawberry wilt from field samples

Verticillium dahliae Kleb, the cause of Verticillium wilt disease, is a destructive pathogen that leads to severe yield losses in strawberry fields and thus considerable economic damages. Although rapid identification and detection methods are becoming available more, pathogen quantification remains one of the main challenges in the disease management. In this study, a real-time polymerase chain reaction (rtPCR) assay was developed to quantitatively assess V. dahliae abundance directly from affected roots and soil collected from different areas in Estonia. A specific primer pair based on the ribosomal DNA (rDNA) internally transcribed spacer was designed for SYBR Green-based assay. Strawberry plant and soil samples were randomly collected from different areas in Estonia and analyzed for V. dahliae by soil plating technique and rtPCR assay. The assay was specific for V. dahliae so that the minimum detection limit was 0.93?pg?µl^?1 of pathogen DNA and the lowest amount of V. dahliae detected in soil was 10.48?pg?µl^?1 of target DNA corresponding to one microsclerotia per gram of soil. This technique allowed rapid detection and quantification of the pathogen DNA at the picogram level in soils and even in symptomless plants, facilitating the screening of the pathogen in diverse areas. This is the first study about the rtPCR technique being used successfully to assess populations of V. dahliae with high specificity and sensitivity in Estonia strawberry fields. Results of this research can be useful for growers and agricultural organizations to improve available disease management strategies against Verticillium wilt.     

Isolation and identification of boron-accumulating bacteria from contaminated soils and active sludge

Abstract

High levels of boron are toxic to living organisms. The removal of boron from contaminated water is an important way of dealing with this environmental problem. For this purpose, we screened for bacteria that can absorb high levels of toxic boron from the environment. A range of boron-accumulating bacteria was isolated from boron-contaminated soil in Turkey, from uncontaminated soil in Tokyo, Japan, and from active sludge in Tokyo by semi-quantifying the boron accumulation of each single-cultured bacterium. Intracellular boron concentrations ranged from 0.3 to 1.36?nmol?g^?1 dry weight. Nineteen isolates identified by 16S rRNA gene sequence analysis were most closely related to the genera Bacillus, Variovorax, Pseudomonas, Shewanella, Mycobacterium and Rhodococcus. To our knowledge, this is the first study examining a wide range of bacterial strains in terms of boron accumulation.     

Characterization of ammonia oxidizing bacteria associated with weeds in a Japanese paddy field using amoA gene fragments

Abstract

Ammonia oxidizing bacteria (AOB) are important microorganisms in rice paddy field ecosystems because they play a key role in the nitrogen (N) cycle by converting ammonia (NH₃) to nitrite (NO^? ₂). In this study, we investigated AOB associated with three types of weeds in a Japanese paddy field (semi-aquatic Echinochloa oryzicola Vasing, floating Lemna paucicostata Hegelm and submerged Najas graminea Delile) using molecular techniques polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and DNA sequencing targeting ammonia monooxygenase (amoA) gene. This work confirmed that rice paddy weeds harbor AOB and that the community composition is different for each type of weed. However, all AOB sequences associated with the tested weeds were closely related to known species of Nitrosospira-like AOB isolated from soil, suggesting that AOB associated with weeds were not specific to weeds and can also be found in the soil. Nitosomanas-like AOB were not detected on any of the weeds tested. In addition, the most dominant AOB strains present in the tested weeds were closely related to Nitrosospira sp. Ka3 and Nitrosospira sp. CT2F. The phylogenetic tree revealed that most of the AOB detected in the present study belonged to amoA cluster 1.     

Purification and isotopic signatures (<Emphasis Type="Italic">δ</Emphasis><Superscript>13</Superscript>C, <Emphasis Type="Italic">δ</Emphasis><Superscript>15</Superscript>N, Δ<Superscript>14</Superscript>C) of soil extracellular DNA

The aim of this work was to obtain pure extracellular DNA molecules so as to estimate their longevity in soil by an isotope-based approach. Extracellular DNA molecules were extracted from all horizons of a forest soil and purified by the procedure of Davis (Purification and precipitation of genomic DNA with phenol–chloroform and ethanol. In: Davis LG, Dibner MD, Battey JF (eds) Basic methods in molecular biology. Appleton & Lange, Norwalk, 16–22, 1986) without (DNA1) or with (DNA2) a successive treatment with binding resins followed by elution. The two differently purified DNA samples were compared for their A₂₆₀/A₂₈₀ ratio, polymerase chain reaction (PCR) amplification and natural abundance of stable (¹³C and ¹⁵N) and radioactive (¹⁴C) isotopes. The purity index and the PCR amplification did not differentiate the efficiency of the two purification procedures. The isotopic signature of DNA was more sensitive and was strongly affected by the purification procedures. The isotopic measurements showed that the major contaminant of extracellular DNA1 was the soil organic matter (SOM), even if it is not possible to exclude that the similar δ ¹³C, δ ¹⁵N and Δ¹⁴C values of DNA and SOM could be due to the use of SOM-deriving C and N atoms for the microbial synthesis of DNA. For extracellular DNA2, extremely low values of Δ¹⁴C were obtained, and this was ascribed to the presence of fossil fuel-derived substances used during the purification, although in amounts not revealed by gas chromatography-mass spectrometry analysis. The fact that it is not possible to obtain contaminant-free DNA molecules and the potential use of soil native organic compounds during the microbial synthesis of DNA make it not achievable to estimate the age of soil extracellular DNA by radiocarbon dating.     

Methods for evaluating human impact on soil microorganisms based on their activity,biomass, and diversity in agricultural soils

The present review is focused on microbiological methods used in agricultural soils accustomed to human disturbance. Recent developments in soil biology are analyzed with the aim of highlighting gaps in knowledge, unsolved research questions, and controversial results. Activity rates (basal respiration, N mineralization) and biomass are used as overall indices for assessing microbial functions in soil and can be supplemented by biomass ratios (C : N, C : P, and C : S) and eco‐physiological ratios (soil organic C : microbial‐biomass C, qCO₂, qN_min). The community structure can be characterized by functional groups of the soil microbial biomass such as fungi and bacteria, Gram‐negative and Gram‐positive bacteria, or by biotic diversity. Methodological aspects of soil microbial indices are assessed, such as sampling, pretreatment of samples, and conversion factors of data into biomass values. Microbial‐biomass C (µg (g soil)^–1) can be estimated by multiplying total PLFA (nmol (g soil)^–1) by the F_PLFA‐factor of 5.8 and DNA (µg (g soil)^–1) by the F_DNA‐factor of 6.0. In addition, the turnover of the soil microbial biomass is appreciated as a key process for maintaining nutrient cycles in soil. Examples are briefly presented that show the direction of human impact on soil microorganisms by the methods evaluated. These examples are taken from research on organic farming, reduced tillage, de‐intensification of land‐use management, degradation of peatland, slurry application, salinization, heavy‐metal contamination, lignite deposition, pesticide application, antibiotics, TNT, and genetically modified plants.     

Short‐term potassium release and fixation in some calcareous soils

Potassium (K⁺) directly released from primary K‐bearing minerals can contribute to plant nutrition. The objective of this research was to assess short‐term K⁺ release and fixation on a range of intensively cropped calcareous soils. Potassium sorption and desorption properties and the contributions of exchangeable‐K⁺ (EK) and nonexchangeable‐K⁺ (NEK) pools to K⁺ dynamics of the soil‐solution system was measured using a modified quantity‐to‐intensity (Q : I) experiment. Release and fixation of K⁺ were varied among soils. The relation between the change in the amount of NEK during the experiment and the initial constrain was linear, and soil ability for K⁺ release and fixation (β) for all soils varied from 0.041 to 0.183, indicating that 4% to 18% of added K⁺ converted to NEK when fixation occurred. The equilibrium potential buffering capacity (PBC) for K⁺ derived from Q : I experiments had significant correlation (r = 0.75, p < 0.01) with β, indicating that PBC depends not only on exchange properties but also on release and fixation properties. The depleted soils showed higher β value than the other soils, indicating much of the added K⁺ was converted to NEK in case of positive constraint. The range of the amount of EK which was not in exchange equilibrium with Ca (E_min) in the experimental conditions was large and varied from 0.68 to 9.00 mmol kg^–1. On average, E_min amounted to 64% of EK. This fraction of EK may not be available to the plant. The parameters obtained from these short‐term K⁺ release and fixation experiments can be used in plant nutrition.     

Adsorption of pure and dirty bacterial DNA on clay minerals and their transformation frequency

Several studies have investigated the adsorption of pure DNA on soil particles and its transformation ability. However, the presence of not purified (dirty) rather than pure DNA is more common in the extracellular soil environment. For this reason, we have investigated the adsorption and binding of dirty DNA on montmorillonite (M) and kaolinite (K) and their transforming ability in comparison to pure DNA. After lysis of Bacillus subtilis cells, induced by KCl, dirty DNA was characterized by the presence of cellular wall debris (cwd) and other constitutional organic components (coc). The dirty DNA was then divided into two fractions, one with cellular wall debris (DNA +cwd) and the other without cellular wall debris (DNA −cwd). B. subtilis BD 1512 (Cm^r) and BD 170 (Cm^s) were selected as donor and recipient bacteria, respectively, for adsorption and transformation studies. Both cwd and coc seem to facilitate the adsorption of DNA to clay minerals, whereas only cwd promote the DNA binding on clays, protecting also the DNA fragments below 400 bp against nucleases. Both dirty DNA fractions adsorbed and bound on clay minerals were able to transform competent cells.     

Measurement of protein synthesis by soil bacterial assemblages with the leucine incorporation technique

The incorporation of [¹⁴C]-leucine into protein by soil organisms was measured both in soil slurries and for bacteria extracted from soil by homogenization-centrifugation. The result was compared with thymidine incorporation. Using a soil slurry, 9.1×10^-10 mol leucine h^-1g^-1 dry weight of soil was incorporated into protein, with a calculated leucine: thymidine ratio (mol:mol) of about 34. Non-specific labelling of macromolecules other than protein was observed with both the soil slurry and the homogenization-centrifugation method. With the latter, 46.5% of the total incorporation was found in the protein fraction (hot-acid insoluble). The incorporation of leucine was linear with time for at least 4 h for extracted bacteria. Even at 2000 nM, [¹⁴C]-leucine did not saturate incorporation into protein. Isotope dilution plots indicated that with 750 nM leucine, the degree of participation of the labelled substance in protein synthesis was 0.59. With this value, the ratio of leucine:thymidine incorporation into total macromolecules was calculated as 41 for extracted bacteria. On the basis of incorporation into protein (leucine) and incorporation into DNA (thymidine) only, the leucine:thymidine ratio was calculated as 117. The mean turnover time of bacteria at 22°C, calculated using conversion factors from published studies and leucine incorporation into protein of extracted bacteria, was 4.3 days.     

Electrokinetic-enhanced remediation of actual arsenic-contaminated soils with approaching cathode and Fe0 permeable reactive barrier

Purpose

The aim of this study was to investigate the remediation efficiency of actual arsenic-contaminated soils by electrokinetic (EK)-enhanced remediation with approaching cathode and Fe⁰ permeable reactive barrier (PRB).

Materials and methods

Experiments were conducted in a lab-made apparatus consisting of the anode reservoir, the soil specimen chamber, and the cathode reservoir.

Results and discussion

In this study, the enhanced combination methods (approaching cathode and Fe⁰-PRB) were assisted for EK remediation of actual arsenic-contaminated soils under a voltage gradient of 1 V/cm and a treatment period of 96 h. Experimental results showed that arsenic accumulated in the anode sections (I, II) of the soil by employing EK alone with an arsenic removal rate of less than 5%. In contrast, EK-enhanced remediation with either approaching cathode (EK/AC) or Fe⁰-PRB (EK/PRB) reduced the arsenic concentrations in both central and anode sections of the soil and afforded the removal rates of 20% in both cases. However, EK-enhanced remediation with the combination of approaching cathode and Fe⁰-PRB (EK/PRB/AC) reached the removal efficiency of 45% without arsenic accumulation in any soil sections. This phenomenon is mainly caused by the approaching cathode that creates an alkaline environment to promote the migration of arsenic, as well as PRB filled with Fe⁰ that achieves the adsorption and immobilization of arsenic.

Conclusions

The highest remediation efficiency was achieved in the EK/PRB/AC test, which was attributed to the fact that the combination of this two methods solved the problem of arsenic accumulation in treated soil and ensured a more thorough arsenic removal. Furthermore, enhanced remediation efficiency does not elevate the costs.

     

Isolation of pentachlorophenol decomposing bacteria from soil

The bacteria capable of degrading pentachlorophenol (PCP) were isolated from soil. In the soil perfused with 40 ppm PCP solution, PCP was decomposed and five chlorine atoms of PCP were liberated as chloride ion after about 3 weeks. Re-addition of PCP after its degradation, accelerated the rate of PCP degradation and de-chlorination. After the addition of PCP to the soil three times, bacteria which grew on PCP agar were counted to be about 2 × 10⁷ per gram dry soil. In the liquid medium inoculated with the perfused soil, PCP degradation and complete de-chlorination were found. In this case, multiplication of bacteria capable of growing on PCP agar was found. The bacteria capable of growing on and degrading PCP in the medium with inorganic salts and 40 ppm PCP as a sole source of carbon were isolated from the agar plates for enumeration of the bacteria. From the morphological and physiological properties of the isolated bacteria, the genus of the bacteria was considered to be Pseudomonas or a closely related one. In the culture medium with PCP and inorganic salts, the bacteria degraded PCP and completely de-chlorinated it. The de-chlorination process corresponded approximately to PCP disappearance. Pathways of PCP metabolism are not yet elucidated.     

不同类型水稻土微生物群落结构特征及其影响因素

选取基于我国土壤地理发生分类的不同类型土壤发育的四种水稻土,利用15N2气体示踪法测定生物固氮速率,采用实时荧光定量PCR(Real-time PCR)技术测定细菌丰度,通过16S rRNA基因高通量测序分析微生物群落组成和多样性.结果表明:变形菌门(Proteobacteria)、酸杆菌门(Acidobacteria...     

Quantitative real-time PCR effectively detects and quantifies colonization of sclerotia of Sclerotinia sclerotiorum by Trichoderma spp.

Some members of the fungal genus Trichoderma are able to colonize and destroy sclerotia, the thick-walled resting structures of the soilborne plant pathogenic fungus Sclerotinia sclerotiorum, thus providing a potential means of biological disease control. However, current methods to detect and quantify colonization of sclerotia are labor-intensive, and generally qualitative rather than quantitative in nature. Our objective was to develop quantitative real-time PCR (polymerase chain reaction) methods to detect and measure colonization of sclerotia by Trichoderma spp. Specific PCR primer/probe sets were developed for Trichoderma spp. and for S. sclerotiorum. A total of 180 ITS1 (internal transcribed spacer) and ITS2 sequences from different species in the genus Trichoderma were aligned, and consensus sequences were determined. Six candidate primer sets were based on conserved regions of the consensus sequence, and the specificity of each nucleotide sequence was examined using BLAST (Basic Local Alignment Search Tool; NCBI) software. Each candidate primer set was tested on genomic DNA of T. harzianum strain ThzID1-M3, as well as six different Trichoderma isolates from soil, and on genomic DNA of S. sclerotiorum. The optimum primer/probe set selected, TGP4, successfully amplified genomic DNA of all Trichoderma isolates tested, and showed high precision and reproducibility over a linear range of eight orders of magnitude, from 85 ng to 8.5 fg of T. harzianum genomic DNA. TGP4 did not amplify S. sclerotiorum DNA. A specific PCR primer/probe set (TMSCL2) was developed for S. sclerotiorum, based on the calmodulin gene sequence. TMSCL2 did not amplify Trichoderma DNA. Quantitative real-time PCR with the primers then was evaluated in experiments to test differential effects of two soil moisture levels (−50 kPa, −1000 kPa matric potential) on biocontrol of S. sclerotiorum by indigenous Trichoderma spp. Periodically over 40 days, Trichoderma and S. sclerotiorum DNA in sclerotia were quantified by PCR with appropriate primers. Over 90% of the sclerotia were colonized by indigenous Trichoderma spp. at −1000 kPa, over the 40-day period, compared to only 60% at −50 kPa. In addition to determining incidence of colonization, the PCR method allowed measurement of the extent of sclerotial colonization, which also was significantly greater in the drier soil. Quantitative real-time PCR with the TGP4 primer/probe set provides a sensitive detection and measurement tool to evaluate colonization of sclerotia by Trichoderma spp.     

Determination of free DNA in soils

The concentration of free DNA in soils has to be determined in order to understand the fate and the transport of extracellular DNA. A protocol for the extraction and determination of free DNA was developed. The procedure uses separation steps, i.e. centrifugation and ultra‐filtration. The free dsDNA was stained with PicoGreen® and determined fluorimetrically. Samples from different soils, different soil horizons, soil waters and under different land use systems were analyzed. It was found that in nearly all samples free DNA was detectable. Free DNA concentrations of up to 1950 ng (g dried sample)^—1 could be detected depending on depth, soil type and system of land use.     

The effect of pharmaceutical waste-fungal biomass,treated to degrade DNA,on the composition of eubacterial and ammonia oxidizing populations of soil

The aim of this work was to study variations in the composition of eubacteria and ammonia-oxidizing populations of soil, both determined by denaturing gradient gel electrophoresis (DGGE), after the addition of a pharmaceutical fungal biomass, treated to degrade its DNA. This waste can be used as an amendment. The fungal biomass waste was added at three rates: 0.05, 0.1, and 1% per dry weight of soil. Control soil, without any amendment, was also investigated. Total DNA was extracted, purified, and amplified by using either universal (eubacteria) or specific (amoA) primers. Amplicons were separated by DGGE. Sequencing was also carried out to better assess the diversity of ammonia oxidizing bacteria. Changes in the composition of eubacterial community were detected after 3 days only in the soil treated with the highest dose, while the ammonia oxidizing population responded more promptly (after 1 day) with evident modifications at level of Nitrosolobus like sequences.     

Comparison of chloroform fumigation-extraction, phospholipid fatty acid, and DNA methods to determine microbial biomass in forest humus

Techniques developed to measure microbial biomass in mineral soils may not give reliable results in humus. We evaluated the relationships between three techniques to estimate microbial biomass in forest humus: chloroform fumigation-extraction (CFE), total extractable phospholipid fatty acids (PLFA), and extractable DNA. There was a good relationship between PLFA and CFE (R²=0.96), with a slope slightly different from that previously reported for mineral soils (1 nmol PLFA corresponded to a flush of 3.2 μg C released by fumigation in humus cf. 2.4 μg C in mineral soil). There was no relationship between DNA concentration and the other two measurements of microbial biomass. This may be due, in part, to the high fungal biomass in forest humus, as DNA concentration per unit biomass is much more variable for fungi than bacteria.     

土壤残渣有机质与固定态铵之间的关系

采集了全国不同类型的土壤40个,分析了土壤全氮、有机质、固定态铵、剩余有机质（KOBr处理后的土壤有机质）、残渣有机质（KOBr-HF处理后的土壤有机质）含量.结果表明,以2∶1型粘粒矿物为主土壤的残渣有机质含量与固定态铵含量之间呈极显著正相关（r=0.831^＊＊）,晶格之间存在的有机质（即残渣有机质与剩余有机质之差值）含量则与固定态铵含量之间也呈极显著正相关（r=0.832^＊＊）,而以1∶1型粘粒矿物为主土壤的残渣有机质含量和晶格有机质含量与固定态铵含量不相关;土壤剩余有机质、残渣有机质的含量分别为2.59 g kg^-1、3.70 g kg^-1,分别占土壤有机质的10%和16%.土壤残渣C/N比（平均值为16.69）明显高于原土壤（平均值为5.37）.     

Diversity of denitrifying microflora and ability to reduce N2O in two soils

 The ozone-depleting gas N₂O is an intermediate in denitrification, the biological reduction of NO₃ ^– to the gaseous products N₂O and N₂ gas. The molar ratio of N₂O produced (N₂O/N₂O+N₂) varies temporally and spatially, and in some soils N₂O may be the dominant end product of denitrification. The fraction of NO₃ ^–-N emitted as N₂O may be due at least in part to the abundance and activity of denitrifying bacteria which possess N₂O reductase. In this study, we enumerated NO₃ ^–-reducing and denitrifying bacteria, and compared and contrasted collections of denitrifying bacteria isolated from two agricultural soils, one (Auxonne, soil A) with N₂O as the dominant product of denitrification, the other (Chalons, soil C) with N₂ gas as the dominant product. Isolates were tested for the ability to reduce N₂O, and the presence of the N₂O reductase (nosZ)-like gene was evaluated by polymerase chain reaction (PCR) using specific primers coupled with DNA hybridization using a specific probe. The diversity and phylogenetic relationships of members of the collections were established by PCR/restriction fragment length polymorphism of 16s rDNA. The two soils had similar numbers of bacteria which used NO₃ ^– as a terminal electron acceptor anaerobically. However, the soil A had many more denitrifiers which reduced NO₃ ^– to gaseous products (N₂O or N₂) than did soil C. Collections of 258 and 281 bacteria able to grow anaerobically in the presence of NO₃ ^– were isolated from soil A and soil C, respectively. These two collections contained 66 and 12 denitrifying isolates, respectively, the others reducing NO₃ ^– only as far as NO₂ ^–. The presence of nosZ sequences was generally a poor predictor of N₂O reducing ability: there was agreement between the occurrence of nosZ sequences and the N₂O reducing ability for only 42% of the isolates; 35% of the isolates (found exclusively in soil A) without detectable nosZ sequences reduced N₂O whereas 21% of the isolates carrying nosZ sequences did not reduce this gas under our assay conditions. Twenty-eight different 16S rDNA restriction patterns (using two restriction endonucleases) were distinguished among the 78 denitrifying isolates. Two types of patterns appeared to be common to both soils. Twenty-three and three types of patterns were found exclusively among bacteria isolated from soils A and C, respectively. The specific composition of denitrifying communities appeared to be different between the two soils studied. This may partly explain the differences in the behaviour of the soils concerning N₂O reduction during denitrification. Received: 31 October 1997     

A new method for determining the metabolic activity of specific bacterial populations in soil using tritiated leucine and immunomagnetic separation

 A new assay, using immunomagnetic separation and uptake of tritiated leucine ([³H]-Leu), was developed for measuring the in situ metabolic activity of specific bacterial populations in soil. Such assays are needed to assess the role individual species play in diverse microbial soil communities. The method was optimized using Pseudomonas putida KT2440 : :Tc⁺/TOL::gfp inoculated into soil microcosms. Inoculated soil samples were incubated with [³H]-Leu followed by an immunomagnetic separation to recover the target bacteria. Radiolabel incorporated by the target bacteria was then measured. Incubation time with immunomagnetic beads was not critical for optimal target cell recovery, but samples needed to be washed at least 5 times during the immunomagnetic separation to reduce unspecific binding of the indigenous soil bacteria to the magnetic beads. Soil absorption of the polyclonal antibody further reduced this unspecific binding, resulting in <1% contamination by indigenous soil bacteria relative to numbers of recovered target cells. The assay was tested by investigating the effect of different incubation temperatures on the metabolic activity of the target cells. As expected, a linear relationship between activity and temperature was observed, demonstrating the sensitivity of the assay. The method was applied to compare activities of the target strain in bulk soil and in the rhizosphere of barley. Contrary to what was anticipated, no significant difference in metabolic activity was observed. Received: 4 November 1999     

不同施肥处理稻田系统磷素输移特征研究

磷是水体富营养化限制性元素,近年来由于磷肥的过量施用,农田迁移的磷素已成为水体磷素的主要来源。本研究通过野外测坑定位试验,研究有机肥处理(OT)、混施肥处理(MT)和化肥处理(CT)3种施肥处理下,稻田中磷素的迁移流失特征及这3种处理对水稻产量和磷素利用率的影响,以探求稻田系统的最佳施磷方式。结果表明,CT、MT和OT 3种施肥方式的磷径流流失负荷分别为0.56 kg(P)·hm-2、1.13 kg(P)·hm-2和4.20 kg(P)·hm-2,渗漏流失负荷分别为0.42 kg(P)·hm-2、0.44 kg(P)·hm-2和0.45 kg(P)·hm-2;磷的径流流失占流失总量的56.86%~90.38%,是水稻田磷素流失的主要途径。磷的径流流失主要受施肥和降雨的影响,50%左右磷的流失发生在第1次径流过程;磷素渗漏流失特征不受施磷处理的影响,80%以上的流失发生在施肥后的前30 d。在磷素流失形态上,坑面水、渗漏水和径流水中磷素的主要形态均为可溶性磷;在土壤方面,MT处理和OT处理能保证土壤磷营养,CT处理的土壤有效磷和有机质含量则显著下降。3种施肥处理的水稻产量显著高于空白对照,且MT最高,为6 728.84 kg·hm-2;磷肥利用率CT和MT处理显著高于OT,CT和MT间差异不显著。综合比较,混施肥处理在磷素流失、土壤养分利用和水稻产量等方面更符合我国生态农业发展的要求。