Detection of microorganisms in undisturbed soil by combining fluorescence in situ hybridization (FISH) and micropedological methods

In contrast to conventional approaches molecular microbiology leads to a deeper understanding of the biodiversity of soil microorganisms. Nevertheless, there is a lack of knowledge regarding the spatial distribution of microbiota in the complex soil matrix and the interaction between the soil structure and microorganisms. DNA analytical methods such as fluorescence in situ hybridization (FISH) are being utilized to improve the characterization of microbial biocoenosis.Micropedological procedures which preserve the soil structure by embedding it with resin, in combination with FISH, allow the localization and identification of soil microorganism diversity in relation to the specific properties of their microhabitats. In this study, FISH was used prior to resin embedding in undisturbed soil samples of four different soils.The polished sections provided visualization of the bound probes as well as the undisturbed soil matrix via fluorescence microscopy. Furthermore, cell counts of active bacteria, locating of hot spots and their relationship to microsites rich in nutrients and water, such as humus or clay minerals, are now easy to perform. This will lead to a better understanding of how soil structure can affect soil microorganisms and vice versa.Derived from the use of 16S rRNA targeted oligonucleotide probes, EUB338 and NON338, the cell counts of FISH-detected bacteria were in the same order of magnitude in the undisturbed and the suspended soil samples. Counterstaining with DAPI showed varying detection rates caused by differing activities of the soil microorganisms.     

Application of rRNA-based probe for detection of a bacterium in rhizoplane

Recently, in situ hybridization technique targeted to ribosomal RNA (Delong et al. 1989) has been widely used to detect specific microorganisms in various environments such as marine environment (Nishimura 1995), sediments (Spring et al. 1993), inside of organisms (Amann et al. 1991), and soil (Hahn et al. 1992). This technique provides information not only on microscale localization of the microorganisms but also on their metabolic activities in situ. So far, detection by in situ hybridization targeted to ribosomes in soil has been scarcely reported because two problems arise when this method is applied to soil. The first is that cells in natural soils cannot be easily stained because the cellular ribosome content is low in an oligotrophic soil environment. The second is that non-specific binding of the probes to soil particles and autofluorescence of soil components such as organic matter and mineral particles interfere with the signal from the probe specifically hybridized.     

From Media to Molecules: New Approaches to the Detection of Micro-organisms in Water

Present methods for the detection of micro-organisms in the environment are slow, inefficient and often unreliable. Alternative approaches which are reliable and rapid, enabling results to be obtained within one working day, are required. The development of molecular techniques, in particular in situ hybridisation offers the potential for rapid and specific assays. This paper describes the use of oligonucleotide probes targeted against the 16S and 18S ribosomal RNA molecules of Escherichia coli and Cryptosporidium parvum for rapid and specific detection. In situ hybridisations with biotinylated peptide nucleic acid (PNA) probes in combination with the tyramide signal amplification (TSA) system enabled the specific detection of Escherichia coli and Cryptosporidium parvum within 3 hours. The C. parvum assay provided a species-specific alternative to the currently available fluorescent antibody approach.     

荧光原位杂交技术检测土壤中博德特氏菌探针的设计与应用

根据博德特氏菌(Bordetellasp.)的16S rRNA基因序列,设计荧光原位杂交(FISH)检测博德特氏菌的寡核苷酸探针FW_iso_62和FW_iso_761,在20%～60%甲酰胺均有很强的荧光信号。采用探针FW_iso_62及其竞争探针,结合Nycodenz和DAPI技术,建立定量检测土壤中博德特氏菌的DAPI-FISH方法。该方法可排除土壤颗粒的自动荧光对细菌信号的掩盖,保证图片中有大量微生物供统计分析,还能有效保存微生物的原位信息。应用该方法分析土壤中1,2,4-三氯苯降解菌-博德特氏菌,结果未受氯苯污染的农田土壤中没有检测到博德特氏菌,而氯苯污染土壤中检测到大量的博德特氏菌,每克土壤含3.78×106个。将该污染土壤中分离的博德特氏降解菌及其降解菌群接种至农田土壤中,降解菌的数量均随培养而增加,一个月后分别占DAPI计数的1.7%和3.8%。本研究设计的探针可有效用于复杂环境样品中博德特氏菌的定性与定量检测。     

Evaluation of the atzB gene as a functional marker for the simazine-degrading potential of an agricultural soil

The occurrence of natural simazine-degrading bacteria could be an important limiting factor to the use of the herbicide in those agricultural soils with a wide history of herbicide applications. In this work the potential of an agricultural soil to degrade simazine and the effect of the addition of urea was assayed in both fertilised and unfertilised soil microcosms. A culture-independent approach based on the fluorescence in situ hybridisation (FISH) technique, using a specific oligonucleotide probe (AtzB1), was applied to detect simazine-degrading bacteria in the soil microcosms. The presence of the atzABC genes in the agricultural soil was confirmed by PCR from soil-extracted DNA. The percentage of AtzB1 probe-target cells in the urea-untreated soil was higher than in the urea-treated one. Moreover, the greatest percentage of AtzB1 probe-target cells in the urea-untreated soil was accompanied by a greater degradation rate, compared to the urea-fertilised one. Our results indicate that the proposed approach was sensitive enough to detect changes in the natural simazine degradation capacity of the soil after fertilisation practices, which typically involve a nitrogen increase.     

Fluorescence detection of aluminum in arbuscular mycorrhizal fungal structures and glomalin using confocal laser scanning microscopy

Arbuscular mycorrhizal spores and glomalin-related soil protein (GRSP) isolated from acid soils were analyzed using confocal laser scanning microscopy (CLSM) for Al detection. Mycorrhizal structures of Glomus intraradices produced under in vitro conditions as well as spores and GRSP from neutral and Cu-polluted soils were used as contrasting criteria. Spores and GRSP from soils with 7 and 70% Al saturation showed autofluorescence which increased especially at the highest soil Al level and when Al³⁺ solution was added. G. intraradices spores showed fluorescence only when exogenous Al³⁺ was added. On the contrary, spores and GRSP from neutral and Cu-polluted soils showed little or no significant fluorescence. This fluorescence shown by fungal structures and GRSP when subjected to high Al (of endogenous or exogenous origin) suggest a high capacity for Al immobilization, which could be an effective way to reduce Al activity and phytotoxicity in acid soils.     

Fluorescence microscopy for visualization of soil microorganisms—a review

Direct microscopic observation of microorganisms is an important tool in many microbial studies. Such observations have been reported for Protozoa, fungi, inoculated bacteria, and rhizosphere microorganisms but few studies have focused on indigenous bacteria and their spatial relationship within various microhabitats. Principles and applications of epifluorescence microscopy and confocal laser scanning microscopy for visualization of soil microorganisms in situ are reviewed. Both cationic and anionic dyes (also commonly referred to as fluorochromes if they are fluorescent) have been used based on their ability to bind to specific cellular components of microbial cells. Common fluorochromes used for imaging of microbial cells include acridine orange, ethidium bromide, fluorescein isothiocyanate, 5-(4,6-dichlorotriazinyl) aminofluorescein, 4,6-diamidino-2-phenylindole, europium chelate, magnesium salt of 8-anilino-1-naphthalene sulfonic acid, and calcofluor white M2R. Combining fluorescence staining techniques with soil thin section technology allows one to obtain images of microorganisms in situ. Soil texture and the procedures used for resin embedding are important factors affecting the quality of stained soil thin sections. Indeed, general limitations of applying fluorescence microscopy to soil ecological studies are the non-specific binding of dyes to the soil matrix and the autofluorescence of some soil components. The development of fluorescent in situ hybridization and confocal laser scanning microscopy techniques provides new potential for microbial distribution studies.     

Fluorescence detection of aluminum in arbuscular mycorrhizal fungal structures and glomalin using confocal laser scanning microscopy

Arbuscular mycorrhizal spores and glomalin-related soil protein (GRSP) isolated from acid soils were analyzed using confocal laser scanning microscopy (CLSM) for Al detection. Mycorrhizal structures of Glomus intraradices produced under in vitro conditions as well as spores and GRSP from neutral and Cu-polluted soils were used as contrasting criteria. Spores and GRSP from soils with 7 and 70% Al saturation showed autofluorescence which increased especially at the highest soil Al level and when Al³⁺ solution was added. G.intraradices spores showed fluorescence only when exogenous Al³⁺ was added. On the contrary, spores and GRSP from neutral and Cu-polluted soils showed little or no significant fluorescence. This fluorescence shown by fungal structures and GRSP when subjected to high Al (of endogenous or exogenous origin) suggest a high capacity for Al immobilization, which could be an effective way to reduce Al activity and phytotoxicity in acid soils.     

A phylogenetic microarray targeting 16S rRNA genes from the bacterial division Acidobacteria reveals a lineage-specific distribution in a soil clay fraction

We designed an oligonucleotide microarray using probe sequences based upon a phylogenetic analysis of 16S rRNA genes recovered from members of the bacterial division Acidobacteria. A total of 42,194 oligonucleotide probes targeting members of the Acidobacteria division at multiple phylogenetic levels were included on a high-density microarray. Positive control hybridizations revealed a linear relationship between hybridization signal and template concentration, and a substantial decrease in non-specific hybridization was achieved through the addition of 2.5 M betaine to the hybridization buffer. A mean hybridization signal value was calculated for each Acidobacteria lineage, with the resultant lineage-specific hybridization data revealing strong predictive value for the positive control hybridizations. The Acidobacteria phylochip was then used to evaluate Acidobacteria rRNA genes from a Wisconsin soil and within a soil clay fraction. The Acidobacteria hybridization profile revealed the predominance of Acidobacteria subdivisions four and six, and also suggested a decrease in the abundance of subdivision six relative to subdivision four in the soil clay fraction. The change in relative abundance of these subdivisions in a soil clay fraction was supported by data from quantitative PCR. These results support the utility of a phylogenetic microarray in revealing changes in microbial population-level distributions in a complex soil microbial assemblage.     

Functional diversity of soil organisms — a review of recent research activities in Germany

The aim of this review is to provide an overview of recent investigations on the functional diversity of soil organisms and to elucidate whether a combination of different phenotypic and genotypic assessment methods can give new insights into the relation of structural (phylogenetic) and functional diversity of soil microbial and faunal communities. The knowledge of functional gene sequences for the major microbial transformations enables studies of their presence and diversity in soils. The concomitant evaluation of phylogenetic identification and functional activity of even individual microbial cells in situ is now possible using such as fluorescence in situ hybridization and microautoradiography. Studies about microbial‐faunal interactions clarifies the importance of soil organisms for soil processes.     

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     

Methodologically controlled variations in laboratory and field pH measurements in waterlogged soils

We have tested the reliability and consistency of conventional pH measurements made on water‐soil mixtures with respect to sieving, drying, ratio of water to soil, and time of shaking prior to measurement. The focus is on a waterlogged soil where the preservation potential of archaeological artefacts is critical. But the study includes agricultural and forest soils for comparison. At a waterlogged site, laboratory results were compared with three different field methods: calomel pH probes inserted in the soil from pits, pH measurements of soil solution extracted from the soil, and pH profiles using a solid‐state pH electrode pushed into the soil from the surface. Comparisons between in situ and laboratory methods revealed differences of more than 1 pH unit. The content of dissolved ions in soil solution and field observations of O₂ and CO₂ concentrations were used in the speciation model PHREEQE in order to predict gas exchange processes. Changes in pH in soil solution following equilibrium in the laboratory could be explained mainly by CO₂ degassing. Only soil pH measured in situ using either calomel or solid‐state probes inserted directly into the soil was not affected by gas exchange processes. Variations on the order of 0.2–0.5 pH unit in different laboratory methods could not be explained by degassing and seem to be soil‐type specific and strongly influenced by drying and shaking. Further attention should be given to standardization of pH measurements, particularly before pH measurements from different soil types are compared.     

A simple and effective ND-FISH probe design for identifying barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis>) chromosomes

Fluorescence in situ hybridization (FISH) has been widely used to analyze the physical mapping of chromosomes and study the chromosome rearrangements during evolution. The conventional FISH technique is time-consuming and labor-intensive as it involves many experimental steps including (1) PCR amplification of the target sequence, (2) recovery of the amplified product, (3) plasmid extraction, (4) fluorescence labeling, as well as (5) denaturing of chromosomes and probes. Here we report an effective method for the design of oligonucleotide probes and the development of a new oligonucleotide probe for non-denaturing FISH in barley. The probe Oligo-442A01 with 6133 copies in barley genome could be mapped to the subtelomeres of barley. However, there were significant differences in signal positions and strengths between Oligo-442A01 and the contrast probe Oligo-HvT01.1, especially on chromosome 3H. These oligonucleotide probes combinations (AGG)₅ and Oligo-442A01, (CTT)₅ and Oligo-HvT01.1 could effectively differentiate the barley chromosomes.     

Organic and synthetic fertility amendments influence soil microbial,physical and chemical properties on organic and conventional farms

Field experiments were conducted to examine the effects of organic and synthetic soil fertility amendments on soil microbial communities and soil physical and chemical properties at three organic and three conventional vegetable farms in Virginia and Maryland in 1996 and 1997. Two treatments, including either an alternative organic soil amendment (composted cotton-gin trash, composted yard waste, or cattle manure) or synthetic soil amendment (fertilizer) were applied to three replicated plots at each grower field location. Production history and time affected propagule densities of Trichoderma species which remained higher in soils from organic farms. Propagule densities of Trichoderma species, thermophilic microorganisms, and enteric bacteria were also detected in greater numbers in soils amended with alternative than synthetic amendments, whereas propagule densities of Phytophthora and Pythium species were lower in soils amended with alternative than synthetic fertility amendments. Concentrations of Ca, K, Mg, and Mn were higher in soils amended with alternative than synthetic fertility amendments. Canonical correlations and principle component analyses indicated significant correlation between these soil chemical factors and the biological communities. First-order canonical correlations were more negative in fields with a conventional history, and use of synthetic fertilizers, whereas canonical correlations were more positive in fields with a history of organic production and alternative soil amendments. In the first year, yields of corn or melon were not different in soil amended with either synthetic or organic amendments at four of six farms. In the second year, when all growers planted tomatoes, yields were higher on farms with a history of organic production, regardless of soil amendment type. Alternative fertility amendments, enhanced beneficial soil microorganisms reduced pathogen populations, increased soil organic matter, total carbon, and cation exchange capacity (CEC), and lowered bulk density thus improving soil quality.     

The structure of fungal biomass and diversity of cultivated micromycetes in Antarctic soils (progress and Russkaya Stations)

The distribution of the fungal biomass and diversity of cultivated microscopic fungi in the profiles of some soils from East (Progress Station, valleys of the Larsemann Hills oasis) and West (Russkaya Station, the Marie Byrd Land) Antarctica regions were studied. The structure of the biomass (spore/mycelium and live cells/dead cells) was analyzed by fluorescence microscopy with staining using a set of coloring agents: calcofluor white, ethidium bromide, and fluorescein diacetate. The species composition of the cultivated microscopic fungi was determined on Czapek’s medium. The fungal biomass in the soils studied is not high (on the average, 0.3 mg/g of soil); the greatest biomass (0.6 mg/g) was found in the soil samples with plant residues. The fungal biomass is mainly (to 70%) represented by small (to 2.5 μm) spores. About half of the fungal biomass is composed of living cells. There are differences in the distribution of the fungal biomass within the profiles of different primitive soils. In the soil samples taken under mosses and lichens, the maximal biomass was registered in the top soil horizons. In the soils with the peat horizon under stone pavements, the greatest fungal biomass was registered in the subsurface horizons. Thirty-eight species of cultivated microscopic fungi were isolated from the soils studied. Species of the genus Penicillium and Phoma herbarum predominated.     

Microbial complexes of hydromorphic soils in the Selenga River delta (Baikal region)

The number and the biomass of microorganisms were determined in the soils of the floodplain and islands in the Selenga River delta. The population of fungi in the soils studied was low. The number of saprotrophic prokaryote microorganisms varied from 10⁶–10⁸ CFU/g of soil in the upper horizons to 10⁴–10⁵ CFU/g in the lower horizons of the soils. This pattern is typical for most zonal soils. The microbial biomass in the floodplain soils was 2–4 times as high as that in the soils of the islands. The number of microorganisms of different ecologic-trophic groups participating in the nitrogen and carbon mobilization was much lower than that in the hydromorphic soils of the Transbaikal region or in the cryogenic soils of the Angara River basin (Irkutsk district). The low coefficient of microbiological mineralization and the low coefficient showing the lack of nitrogen (coefficient of oligotrophness) in the soils indicated the weak processes of organic matter decomposition in the soils studied. During the season investigated (August–September), the bacterial complexes in all the soils were dominated by bacteria of the genera Bacillus, Pseudomonas, and Aquaspirillum. In the floodplain soils, streptomycetes constituted a considerable part of the microbial complexes of the floodplain soils, whereas, in the soils of the islands, their number was minor.     

Identification and localization of food-source microbial nucleic acids inside soil nematodes

Microorganisms (e.g., prokaryotes, fungi) are food sources for soil nematodes, but they can also be potential mutualists or pathogens. Understanding the linkages between microorganism and invertebrate diversity in soils requires the ability to distinguish between these microbial roles. We tested the potential of a taxon-specific fluorescent in situ hybridization (FISH) procedure for identifying and localizing microbial rRNA within the bodies of soil nematodes. Our objective was to determine whether the rate of digestion permitted detection and identification of food-source nucleic acids within the nematode digestive system (i.e., pharynges, intestines) before their breakdown. First, using laboratory cultures of Caenorhabditis elegans maintained on Escherichia coli, we were able to localize bacterial rRNA throughout the nematode pharynx with the universal bacterial-probe EUB338, although never in the intestines. Second, we applied the fungal rRNA probe FR1 to Aphelenchus avenae cultured on the fungus Rhizoctonia solani. We were unable to detect fungal rRNA within these nematodes, and it appears that this material may be digested rapidly. Next, we applied our technique to nematodes extracted directly from soils. We were able to localize bacterial rRNA within the pharynges of bacterial-feeding species of nematodes from desert soils. We also localized archaeal rRNA using the probe ARC344. Finally, application of EUB338 to desert soil nematodes revealed the presence of bacteria in the intestines of some nematodes and within the ovary of a single nematode. This technique has great potential for use in understanding the feeding behavior of bacterial-feeding soil nematodes and in studies of nematode:bacterial relationships.     

Autolysis of chlamydospores of Fusarium solani f. sp. cucurbitae in chitin and laminarin amended soils

Lysis of chlamydospores of Fusarium solani f. sp. cucurbitae is enhanced both in chitin and in laminarin amended soils. In soil amended with both chitin and laminarin, lysis of chlamydospores resembles that in control soil. Addition of chitin and laminarin to soil stimulates the growth of both bacteria and actinomycetes. In soils amended with chitin and with both chitin and laminarin, a chitinolytic microflora is stimulated. Penetration of chlamydospore cells by soil microorganisms has not been observed using the electron microscope. The mechanism of lysis of chlamydospores in soil is discussed.     

Changes in soil microbial community structure by Allonychiurus kimi (Collembola) and their interactive effects on glyphosate degradation

Collembolans have been known to be involved in various soil ecosystem functions. However, the role of Collembola in organic contaminant degradation has not been sufficiently elucidated to assess its contribution. In this study, varying densities of Allonychiurus kimi (Lee, 1973) (0, 10, and 30 individuals per 30 g of soil) were introduced into glyphosate-contaminated soils (74.1 mg glyphosate kg⁻¹ soil). This study investigated changes in the microbial community and the residual glyphosate concentration in soils over incubation time to elucidate the effects of A. kimi on the glyphosate degradation through its influence on the microbial community. Furthermore, the investigation was conducted in soils collected in May and September 2018 to assess the contribution of A. kimi to glyphosate degradation in soils with varying microbial compositions and biomass. Autoclaved soil was used as a control to minimize the influence of indigenous soil microorganisms on glyphosate degradation. We hypothesize that as the initial density of A. kimi increases, the effects of A. kimi on the soil microbial community become pronounced, altering the degradation kinetics of glyphosate in the soil. The composition and biomass of the soil microorganisms were quantified using the phospholipid fatty acid (PLFA) method. Our study determined that the presence of A. kimi altered the microbial community structure by increasing the bacterial and total microbial, but not fungal, biomass. After seven days of treatment, the bacterial and total microbial biomass in the treatment with A. kimi were >2.0-fold and 1.5-fold greater, respectively, compared to those in the treatments without A. kimi. Specifically, the concentration of PLFA 18:1ω7c, i15:0, and 16:1ω7c was positively correlated with A. kimi density. The residual glyphosate concentration decreased exponentially over time as A. kimi density increased. At the end of the experiment, the remaining portions (%) of glyphosate in the May soil samples were 26.3, 20.1, and 6.2, with A. kimi densities of 0, 10, and 30 per vessel, respectively, and the portions in the September soil samples were 13.4, 12.7, and 2.2, respectively. The DT₅₀s (time required for 50 % degradation) decreased significantly with increasing A. kimi density, ranging from 6.8 to 10.1 days at an A. kimi density of 30 to 12.9–19.4 days without A. kimi. However, in the autoclaved soil, a similar effect was not apparent (i.e., DT₅₀s ranged from 23.3 to 27.4 days). Our study demonstrated that Collembola can enhance organic contaminant degradation in soils by altering the microbial community structure.     

A method for linking in situ activities of hydrolytic enzymes to associated organisms in forest soils

A root window-based, enzyme-imprinted, membrane system has been modified to enable visualization of the activities of hydrolytic enzymes (acid phosphatase, aminopeptidase, chitinase, and β-glucosidase) in situ in forest soils. The approach can be used to correlate the distribution of enzyme activity with visible features such as roots, mycorrhizas, or mycelial mats. In addition, it enables accurate spatial soil sampling for analysis of microbial communities associated with enzyme activities. The substrates are colorimetric conjugates of napthol, where color develops instantly in the field, or fluorimetric conjugates of 4-methylumbelliferone, whose fluorescent products are detected by a gel-documenting system. The method will allow important questions about the relationship between taxonomic and functional diversity of soil microorganisms to be addressed and identification of enzyme activity hot-spots in soil.