Evaluation of the selective respiratory inhibition method for measuring the ratio of fungal:bacterial activity in acid agricultural soils

A procedure for the measurement of the fungal and bacterial contribution to substrate-induced respiration was tested in three arable soils. Glucose and different amounts of cycloheximide (eukaryote inhibitor) and streptomycin sulfate (prokaryote inhibitor) were added to soil suspensions, and respiration (CO₂ evolution) was measured. Streptomycin sulfate concentrations from 10 to 120 mg ml^–1 soil solution caused a stable inhibition of respiration. Amounts of cycloheximide ranging from 5 to 35 mg ml^–1 showed an increasing inhibition. In a test with separate and combined addition of the antibiotics at maximum inhibitory concentrations, inhibition by streptomycin was completely overlapped by cycloheximide. This indicated non-target inhibition which may lead to overestimation of fungal respiration. Experiments with sterilized soils inoculated with either fungi or bacteria confirmed that streptomycin selectively inhibited bacteria. Cycloheximide, however, did not only inhibit fungal respiration already at 2 mg ml^–1, but also increasingly inhibited bacterial respiration at increasing concentrations. Only at less than 5 mg cycloheximide ml^–1 was the condition of selective fungal inhibition fulfilled. When 2 mg cycloheximide and 10 mg streptomycin sulfate ml^–1 were applied, the sum of the separate inhibitions almost equalled the combined inhibition by the mix of both inhibitors in field samples. This method yielded fungal:bacterial respiration ratios of 0.50 to 0.60, and confirmed the dominance of bacteria in Dutch arable soils. The ratios obtained by the selective inhibitors were not correlated with, and were higher than, ratios of fungal:bacterial biovolume (0.19 to 0.46) as determined by microscopy and image analysis. Similar measurements in a forest soil (A-horizon) raised doubts on the reliability of the fungal inhibition by cycloheximide in this soil. It is concluded that the separate:combined inhibition ratio should always be checked, and comparison with other approaches is recommended. Received: 17 September 1996     

15N-氨基糖探针技术新用法:区分和量化土壤真菌和细菌固持无机氮速率

土壤微生物对无机氮的固持作用是构成土壤保氮机制的重要组成。作为土壤微生物的两大主要类群,真菌和细菌是微生物固持无机氮作用的主要参与者。然而,由于土壤微生物的高度复杂多变性,如何有效区分和量化土壤中真菌和细菌各自对无机氮的固持作用是个难题。针对该问题,本文采用“氨基糖稳定同位素探针（AS-SIP）”技术来区分和表征土壤中真菌、细菌各自对无机氮的固持速率。基于此进一步揭示了农业利用和外源碳输入分别对土壤真菌、细菌各自固持硝态氮作用的影响及原因,构建了土壤中真菌、细菌各自固持无机氮实际速率的估算模型,为区分和量化土壤中真菌、细菌各自对无机氮的实际固持速率提供了更为可信的新方法。本文介绍了AS-SIP 技术原理、主要技术优势、应用案例、不足之处以及改进对策,以期推进该方法的应用和发展。     

Quantitative assessment of the fungal contribution to microbial tissue in soil

The fungi-to-bacteria ratio in soil ecological concepts and its application to explain the effects of land use changes have gained increasing attention over the past decade. Four different main approaches for quantifying the fungal and bacterial contribution to microbial tissue can be distinguished: (1) microscopic methods, (2) selective inhibition, (3) specific cell membrane components and (4) specific cell wall components. In this review, the different methods were compared and we hypothesized that all these approaches result in similar values for the fungal and bacterial contribution to total microbial biomass, activity, and residues (dead microbial tissue) if these methods are evenly reliable for the estimation of fungal biomass. The fungal contribution to the microbial biomass or respiration varied widely between 2 and 95% in different data sets published over the past three decades. However, the majority of the literature data indicated that fungi dominated microbial biomass, respiration or non-biomass microbial residues, with mean percentages obtained by the different methodological approaches varying between 35 and 76% in different soil groups, i.e. arable, grassland, and forest soils and litter layers. Microscopic methods generally gave the lowest average values, especially in arable and grasslands soils. Very low ratios in fungal biomass C-to-ergosterol obtained by microscopic methods suggest a severe underestimation of fungal biomass by certain stains. Relatively consistent ratios of ergosterol to linoleic acid (18:2ω6,9) indicate that both cell membrane components are useful indicators for saprotrophic and ectomycorrhizal fungi. More quantitative information on the PLFA content of soil bacteria and the 16:1ω5 content of arbuscular mycorrhizal fungi is urgently required to fully exploit the great potential of PLFA measurements. The most consistent results have been obtained from the analysis of fungal glucosamine and bacterial muramic acid in microbial residues. Component-specific δ¹³C analyses of PLFA and amino sugars are a promising prospect for the near future.     

Effect of Continuous Monocropping of Tomato on Soil Microorganism and Microbial Biomass Carbon

Soil microorganisms play an important role in recycling and transformation of nutrients. Soil microbiological parameters and microbial biomass carbon (MBC) have been suggested as possible indicators of soil quality. Soil microorganisms and MBC in different continuous cropping soils were investigated. Results showed that bacterial population was the highest, followed by actinomycetes, and fungi were the lowest at 0–30 cm soil depth. The amount of soil microorganisms decreased with increasing soil depth (0–10 > 10–20 > 20–30 cm). Soil microbial ratios at different depths proved to be responsive to time (year) variations in continuous monocropping tomato, except those at 0–10 to 10–20 cm depth for fungi and 10–20 to 20–30 cm depth for bacteria. Soil MBC for 12 years of continuous cropping was significantly lower than those for 5, 8, and 10 years (P < 0.05). Continuous cropping years, soil depth, and the interaction of these two parameters significantly influenced soil fungal, bacterial, and actinomycetes populations and MBC. Bacterial population at the 0–10 cm soil layer was a sensitive indicator of continuous cropping of tomato. Soil fungal count increased with increasing monocropping time within 5–8 years.     

Particle size fractionation of fungal and bacterial biomass in subalpine grassland and forest soils

Characterization of soil aggregates according to particle size fractions is a useful tool in process-oriented research into soil organic matter and biological properties. Substrate-induced respiration (SIR) inhibition was used to quantify microbial, fungal and bacterial biomass in particle size fractions of soils ranging from forest to grassland in a subalpine region of central Taiwan. In addition, ergosterol content was determined in the same samples to verify fungal biomass measured by SIR inhibition technique. Surface soil (0–10 cm) was fractionated into four particle size fractions: coarse sand (250–2000 μm), fine sand (53–250 μm), silt (2–53 μm) and clay (0.2–2 μm). The larger sized fractions (>250 μm and 53–250 μm) contained higher levels of fungal ergosterol than the smaller sized ones (2–53 μm and 0.2–2 μm). The largest particle size fraction (250–2000 μm) from all studied habitats showed the highest level of microbial biomass, with no clear trend in microbial biomass level among the other size fractions. SIR-calculated fungal biomass level and ergosterol converted fungal biomass content were positively correlated (r=0.71, p<0.05), and such correlation decreased as biomass levels were high. Ratios of fungi to bacteria ranged between 0.6 and 1.3 in fractions obtained in this study. This study indicates a high variability of microbial (fungal and bacterial) biomass level among particle size fractions in soil, and that the large-sized fractions tend to contain a high level of microbial biomass in a given ecosystem.     

Impact of cattle grazing and inorganic fertiliser additions to managed grasslands on the microbial community composition of soils

A study was undertaken to determine if cattle grazing on managed grasslands had an impact on the microbial community composition of soils. Microbial community molecular profiles of bacteria, actinomycetes, pseudomonads and fungi were generated by polymerase chain reaction (PCR) amplification of rDNA sequences from community DNA isolated from soils. PCR products were profiled using denaturing gradient gel electrophoresis (DGGE) and analysed by principal co-ordinate analysis. PCR–DGGE profiles indicated that cattle grazing had an impact on the pseudomonad community structure only, and that the addition of inorganic nitrogen (N) fertiliser impacted on bacterial, actinomycete and pseudomonad community structure. There was no difference in the community profiles of fungi from grazed and N fertilised grassland plots. Analysis of phospholipid fatty acid (PLFA) profiles revealed that both cattle grazing and N fertiliser impacted on microbial community structure. The abundance of individual PLFAs differed between treatments, with bacterial (15:0), actinomycete (10Me18:0) and fungal (18:2ω6) PLFAs not affected directly by grazing cattle and N fertiliser, however, there were significant grazing–fertiliser interactions. Bacterial plate counts were highest in the N fertilised plots and fungal plate counts were highest in the cattle grazed plots. Analysis of molecular microbial community profiles with PLFA and background soil data revealed several significant correlations. Notably, soil pH was positively correlated with PCO1 of the pseudomonad community profiles and negatively correlated with the fungal PLFA 18:2ω6. Fungal DGGE profiles were negatively correlated with the fungal PLFA 18:2ω6, and bacterial and fungal plate counts positively correlated with each other. Correlation analysis using PC1 from PLFA profile data showed no significant relationship with soil organic matter, pH, total C and total N. The results indicate that cattle grazing and N fertiliser addition to grasslands impact on the community composition of specific groups of micro-organisms. The consequences of such changes in population structure may have implications regarding the dynamics of nutrient turnover in soils.     

A soil microbial community structural-functional index: the microscopy-based total/active/active fungal/bacterial (TA/AFB) biovolumes ratio

In most studies of fungal–bacterial communities in soils, single-value indices such as fumigation–extraction (FE) of microbe-derived organic carbon, measures of specific microbial cell chemical constituents, or activity-related measures have been used. These widely used single value indices, however, do not provide information on the physical structure of the filamentous fungal and bacterial community in a soil. The filamentous fungi, considered as indeterminate organisms, have a variable and changing hyphal network, most of which is devoid of cytoplasm. To meet this need for a direct integrated measure of the physical characteristics of the indeterminate fungi and their associated bacteria, a microscopy-based microbial biovolumes ratios approach is suggested. To provide this information, the total and active biovolumes of both the filamentous fungi and bacteria are assessed by microscopy. To normalize these responses, the ratio of total to active (TA) fungal plus bacterial biovolumes is divided by the ratio of the active fungal to bacterial biovolume (AFB), to yield the total/active/active fungal/bacterial (TA/AFB) biovolumes ratio. This approach has been used to analyze data from recently-cultivated early successional (ES) and uncultivated late successional (LS) sites at a shortgrass steppe of northeastern Colorado, where control plots were compared with those receiving mineral nitrogen amendments, using samples taken during the summer of 1995. The TA/AFB ratio index showed distinct and significant decreases in response to soil disturbance which reflected the decreased hyphal lengths present in these disturbed soils. These changes were not detected by the use of FE-based extractable carbon measurements. The TA/AFB ratio also showed significant positive correlations with indices of plant community development and mineral nitrogen, especially in the plots not amended with N. This TA/AFB ratios index should be able to provide information on the physical structure of the indeterminate filamentous fungi and associated soil bacteria for use in the assessment of soil quality, health and resiliency.     

Do growth yield efficiencies differ between soil microbial communities differing in fungal:bacterial ratios? Reality check and methodological issues

Soil communities dominated by fungi such as those of no-tillage (NT) agroecosystems are often associated with greater soil organic matter (SOM) storage. This has been attributed in part to fungi having a higher growth yield efficiency (GYE) compared to bacteria. That is, for each unit of substrate C utilized, fungi invest a greater proportion into biomass and metabolite production than do bacteria. The assumption of higher fungal efficiency may be unfounded because results from studies in which fungal and bacterial efficiencies have been characterized are equivocal and because few studies have measured microbial GYE directly. In this study, we measured microbial GYE in agricultural soils by following ¹³C-labeled glucose loss, total CO₂-C, and ¹³CO₂-C evolution at 2 h intervals for 20 h in two experiments (differing in N amendment levels) in which the fungal:bacterial biomass ratios (F:B) were manipulated. No differences in efficiency were observed for communities with high versus low F:B in soils with or without added inorganic N. When calculated using ¹³CO₂-C (in contrast to total CO₂-C) evolution, growth yield efficiencies of soils having high and low F:B were 0.69±0.01 and 0.70±0.01, respectively. When soils were amended with N, soils with high and low F:B had growth yield efficiencies of 0.78±0.01 and 0.76±0.01, respectively. Our experiments do not support the widely held assumption that soil fungi have greater growth efficiency than soil bacteria. Thus, claims of greater fungal efficiency may be unsubstantiated and should be evoked cautiously when explaining the mechanisms underlying greater C storage and slower C turnover in fungal-dominated soils.     

Specific antibiotics and nematode trophic groups agree in assessing fungal:bacterial activity in agricultural soil

There are no methods at hand with a long and proven record for assessing the relative contribution of fungi and bacteria to decomposer activity in soil. Whereas a multitude of methods to determine fungal and bacterial biomass are available, activity assays traditionally relied on the substrate-induced respiration (SIR) inhibition approach. Here we compare fungal contribution to the microbial active biomass assessed by the SIR inhibition method with the contribution of fungal-feeding nematodes to the microbial-feeding nematode community. Four cultivation systems on the same soil that differ in carbon inputs with a factor two ranked exactly the same with the two methods. A conventionally farmed rotation with low organic input had the lowest fungal fraction, while three organically farmed soils ranked higher.     

Responses of soil microbial communities to manure and biochar in wheat cultivation of a rice-wheat rotation agroecosystem in East China

Soil contamination in agroecosystems remains a global environmental problem. Biochar has been suggested as an organic amendment to alleviate soil pollution, sequester carbon(C), and improve soil fertility. However, information on how bacterial and fungal communities in acidic bulk and rhizosphere soils respond to swine manure and its biochar is still lacking. In this study, biochar and swine manure were applied at two rates of 1.5 and 3 t ha^-1 in a rice-wheat rotation field to assess ...     

Nutrient limitations to bacterial and fungal growth during cellulose decomposition in tropical forest soils

Nutrients constrain the soil carbon cycle in tropical forests, but we lack knowledge on how these constraints vary within the soil microbial community. Here, we used in situ fertilization in a montane tropical forest and in two lowland tropical forests on contrasting soil types to test the principal hypothesis that there are different nutrient constraints to different groups of microorganisms during the decomposition of cellulose. We also tested the hypotheses that decomposers shift from nitrogen to phosphorus constraints from montane to lowland forests, respectively, and are further constrained by potassium and sodium deficiency in the western Amazon. Cellulose and nutrients (nitrogen, phosphorus, potassium, sodium, and combined) were added to soils in situ, and microbial growth on cellulose (phospholipid fatty acids and ergosterol) and respiration were measured. Microbial growth on cellulose after single nutrient additions was highest following nitrogen addition for fungi, suggesting nitrogen as the primary limiting nutrient for cellulose decomposition. This was observed at all sites, with no clear shift in nutrient constraints to decomposition between lowland and montane sites. We also observed positive respiration and fungal growth responses to sodium and potassium addition at one of the lowland sites. However, when phosphorus was added, and especially when added in combination with other nutrients, bacterial growth was highest, suggesting that bacteria out-compete fungi for nitrogen where phosphorus is abundant. In summary, nitrogen constrains fungal growth and cellulose decomposition in both lowland and montane tropical forest soils, but additional nutrients may also be of critical importance in determining the balance between fungal and bacterial decomposition of cellulose.     

Influence of difloxacin-contaminated manure on microbial community structure and function in soils

Difloxacin (DIF) belongs to the fluoroquinolones, a frequently detected group of antibiotics in the environment. It is excreted in pig manure to a large extent and may consequently reach soils in potentially effective concentrations via manuring. The aim of this study was to assess the effects of DIF-spiked manure on microbial communities and selected functions in soils in a microcosm experiment up to 1 month after application. To test a dose dependency of the effects, three different concentrations of DIF (1, 10 and 100 mg/kg of soil) were used. Microcosms with application of pure manure, as well as untreated microcosms served as control. The addition of pure manure resulted in an increase of microbial biomass and soil respiration as well as a reduced bacteria/fungi ratio. Due to the fast and strong immobilisation of DIF, effects of the antbiotic compound were only visible up to 8 days after application (microbial biomass; respiration; potential denitrification; ratio of bacteria/fungi). As expected these short-term effects resulted in reduced potential denitrification rates as well as a reduced bacteria/fungal ratio in the treatments were DIF has been applied. Surprisingly, microbial biomass values as well as respiration rates were increased by DIF application. Other parameters like nitrate and ammonium content in soil were not influenced by DIF application at any time point. Long-term effects (32 days after application) were only visible for the potential nitrification rates. For those parameters that were influenced by the DIF application a clear dose dependency could not be described.     

The use of phospholipid fatty acid analysis to estimate bacterial and fungal biomass in soil

The cell content of 12 bacterial phospholipid fatty acids (PLFA) was determined in bacteria extracted from soil by homogenization/centrifugation. The bacteria were enumerated using acridine orange direct counts. An average of 1.40×10^-17 mol bacterial PLFA cell^-1 was found in bacteria extracted from 15 soils covering a wide range of pH and organic matter contents. With this factor, the bacterial biomass based on PLFA analyses of whole soil samples was calculated as 1.0–4.8 mg bacterial C g^-1 soil C. The corresponding range based on microscopical counts was 0.3–3.0 mg bacterial C g^-1 soil C. The recovery of bacteria from the soils using homogenization/centrifugation was 2.6–16% (mean 8.7%) measured by PLFA analysis, and 12–61% (mean 26%) measured as microscopical counts. The soil content of the PLFA 18:26 was correlated with the ergosterol content (r=0.92), which supports the use of this PLFA as an indicator of fungal biomass. The ratio 18:26 to bacterial PLFA is therefore suggested as an index of the fungal:bacterial biomass ratio in soil. An advantage with the method based on PLFA analyses is that the same technique and even the same sample is used to determine both fungi and bacteria. The fungal:bacterial biomass ratio calculated in this way was positively correlated with the organic matter content of the soils (r=0.94).     

Interaction between gas exchange rates, physical and microbiological properties in soils recently subjected to agriculture

The impact of conventional tillage (CT) or no-till (NT) management on soil microbial respiration as well as microbial abundance was studied in soils from the El Salado basin river (Buenos Aires, Argentina) recently subjected to agriculture under a corn-pasture rotation since 1996. Both management systems were monitored for several soil (micro)biological, physical and chemical properties during the second (1997) to fourth (1999) years from the beginning of the experiment. O₂ and CO₂ composition of the soil atmosphere and the rate at which soil consumes O₂ (qO₂) or produces CO₂ (qCO₂), under conditions that approximate the soil environment in the field, were quantitated following an experimental method and a mathematical model developed by ourselves [Soil Sci. 166 (2001) 68] to interpret the data. qO₂ and qCO₂ expressed in terms of kg O₂ or CO₂-C per ha per day or per kg C of microbial biomass (microbial respiration), increased from the lowest values measured at 10–30% water-filled pore space (WFPS) up to 60% WFPS, decreasing thereafter. Low respiratory quotients, RQ (qCO₂/qO₂<1.0), were detected, with gas exchanges being slightly higher in NT than in CT. Correspondingly, higher bacterial and fungal biomass were measured in NT than in CT. Apparently, bacteria were more sensitive to high WFPS than fungi. When aerobic bacteria or fungi counts were compared at low or high WFPS, they differed significantly only in the upper soil profile whereas microaerophilic bacteria and fungi were significatively different in both depths tested (D1=5–10 cm; D2=15–20 cm). The results are discussed in terms of microbial metabolism behavior and abundance as a function of management and soil air/water balance in soils recently subjected to agriculture.     

Effect of heptachlor on numbers of bacteria,actinomycetes and fungi in soil

Growth of many soil microorganisms was inhibited on culture media containing heptachlor. At a concentration of 25 mg/l., heptachlor killed 63 per cent of the bacteria transferred from soil dilution plates. Heptachlor, at 100 mg/1. in agar media used for isolating microorganisms from soil, prevented the development of 89 per cent of the bacteria, 81 per cent of the actinomycetes, and 50 per cent of the fungi that appeared on isolation plates without heptachlor. After heptachlor was added to soil, fungal populations declined and bacterial populations increased. Numbers of bacteria were related to amount of heptachlor added; higher concentrations of heptachlor in soil resulted in larger populations. A selective increase in numbers of fungi which would grow on media containing heptachlor at 100 mg/l. occurred in soils amended with heptachlor in amounts ordinarily used in field practices, but a similar increase of heptachlor-resistant bacteria occurred only in soils amended with higher amounts of heptachlor.     

Abundance, production and stabilization of microbial biomass under conventional and reduced tillage

Soil tillage practices affect the soil microbial community in various ways, with possible consequences for nitrogen (N) losses, plant growth and soil organic carbon (C) sequestration. As microbes affect soil organic matter (SOM) dynamics largely through their activity, their impact may not be deduced from biomass measurements alone. Moreover, residual microbial tissue is thought to facilitate SOM stabilization, and to provide a long term integrated measure of effects on the microorganisms. In this study, we therefore compared the effect of reduced (RT) and conventional tillage (CT) on the biomass, growth rate and residues of the major microbial decomposer groups fungi and bacteria. Soil samples were collected at two depths (0-5 cm and 5-20 cm) from plots in an Irish winter wheat field that were exposed to either conventional or shallow non-inversion tillage for 7 growing seasons. Total soil fungal and bacterial biomasses were estimated using epifluorescence microscopy. To separate between biomass of saprophytic fungi and arbuscular mycorrhizae, samples were analyzed for ergosterol and phospholipid fatty acid (PLFA) biomarkers. Growth rates of saprophytic fungi were determined by [¹⁴C]acetate-in-ergosterol incorporation, whereas bacterial growth rates were determined by the incorporation of ³H-leucine in bacterial proteins. Finally, soil contents of fungal and bacterial residues were estimated by quantifying microbial derived amino sugars. Reduced tillage increased the total biomass of both bacteria and fungi in the 0-5 cm soil layer to a similar extent. Both ergosterol and PLFA analyses indicated that RT increased biomass of saprophytic fungi in the 0-5 cm soil layer. In contrast, RT increased the biomass of arbuscular mycorrhizae as well as its contribution to the total fungal biomass across the whole plough layer. Growth rates of both saprotrophic fungi and bacteria on the other hand were not affected by soil tillage, possibly indicating a decreased turnover rate of soil microbial biomass under RT. Moreover, RT did not affect the proportion of microbial residues that were derived from fungi. In summary, our results suggest that RT can promote soil C storage without increasing the role of saprophytic fungi in SOM dynamics relative to that of bacteria.     

Forms of phosphorus in bacteria and fungi isolated from two Australian soils

To understand the origin of organic and condensed forms of phosphorus (P) in soils, detailed information about P forms in microorganisms is required. We isolated 7 bacteria and 8 fungi from two Australian soils and analyzed the P forms in their pure cultures by extraction with NaOH-EDTA followed by ³¹P solution nuclear magnetic (NMR) spectroscopy. The bacteria belonged to the actinobacteria and the fungi to the ascomycota, as determined by rDNA sequencing. The proportions of broad forms of P were significantly different between the bacterial and fungal isolates (analysis of similarities, p = 0.001). Ortho-, pyro- and polyphosphate were present in higher proportions in fungi, while monoester and diester P were present in higher proportions in bacteria. Spectral deconvolution of the monoester region revealed 15 distinct resonances. The three major ones, which were identified by spiking experiments as glycerol 1-phosphate, glycerol 2-phosphate and adenosine-5′-monophosphate (AMP), comprised 56–74% of P in the monoester region. Ordination by principal component analysis and testing for treatment effects using analysis of similarities showed significant separation of P distribution in the monoester region between bacterial and fungal isolates (p = 0.007). However, neither group of microorganisms had a specific single P form which might be considered characteristic. As such, it may be difficult to distinguish soil P from bacterial or fungal origins, with the possible exception of a predominantly fungal origin of pyro- and polyphosphate. The identification of three major resonances in the monoester region of microorganisms is important, since the same resonances are found in ³¹P NMR spectra of soil extracts.     

The fungal and bacterial biomass (selective inhibition) and the production of CO<Subscript>2</Subscript> and N<Subscript>2</Subscript>O by soddy-podzolic soils of postagrogenic biogeocenoses

In the humus horizon of soddy-podzolic soils of postagrogenic cenoses and primary forests, the contributions of the fungi and bacteria were determined by the selective inhibition of the substrate-induced respiration (SIR) by antibiotics; the basal (microbial) respiration and the net-produced nitrous oxide (N₂O) were also determined. The procedure of the SIR separation using antibiotics (cycloheximide and streptomycin) into the fungal and bacterial components was optimized. It was shown that the fungi: bacteria ratio was 1.58, 2.04, 1.55, 1.39, 2.09, and 1.86 for the cropland, fallow, and different-aged forests (20, 45, 90, and 450 years), respectively. The fungal and bacterial production of CO₂ in the primary forest soil was higher than in the cropland by 6.3 and 11.4 times, respectively. The production of N₂O in the soils of the primary and secondary (90-year-old) forests (3 and 7 ng N-N₂O/g soil per hour, respectively) was 2–13 times lower than in the postagrogenic cenoses, where low values were also found for the microbial biomass carbon (C_mic), its components (the C_mic-bacteria and C_mic-fungi), and the portion of C_mic in the organic carbon of the soil. A conclusion was drawn about the misbalance of the microbial processes in the overgrown cropland accompanied by the increased production of N₂O by the soil during its enrichment with an organic substrate (glucose).     

Evaluation of quantitative and qualitative recovery of bacterial communities from different soil types by density gradient centrifugation

Extracting and purifying a representative fraction of bacteria from soil is necessary for the application of many techniques of microbial ecology. Here the influence of different soil types on the quantitative and qualitative recovery of bacteria by soil grinding and Nycodenz density gradient centrifugation was investigated. Three soils presenting contrasted physicochemical characteristics were used for this study. For each soil, the total (AODC: acridine orange direct count) and culturable (cfu: colony-forming units) bacterial densities were measured in three distinct fractions: (i) the primary soil, (ii) the soil pellet (soil remaining after centrifugation), and (iii) the extracted cells. The automated–ribosomal intergenic spacer analysis (A-RISA) was used to characterize the community structure directly from the DNA extracted from each fraction. The physicochemical characteristics of soils were found to influence both the efficiency of bacterial cell recovery and the representativeness of the extracted cells in term of community structures between the different fractions. Surprisingly, the most representative extracted cells were obtained from the soil exhibiting the lowest efficiency of cell recovery. Our results demonstrated that quantitative and qualitative cell recovery using Nycodenz density gradient centrifugation are not necessarily related and could be differentially biased according to soil type.     

Novel antibiotics as inhibitors for the selective respiratory inhibition method of measuring fungal:bacterial ratios in soil

The use of the selective inhibition (SI) method for measuring fungal:bacterial ratios may be limited due to biocide selectivity and the overlap of antibiotic activity. This study evaluated novel pairs of antibiotics for their specificity in soils of different origins and their potential reduction in inhibition of non-target organisms. Four soils selected for this study were from a semi-arid shrub-steppe, a loblolly pine forest and two grassland sites (restored and farmed prairie plots). Three bactericides were tested: oxytetracycline hydrochloride, streptomycin sulphate, and bronopol. Three fungicides were tested: captan, ketoconazole, and nystatin. The inhibitor additivity ratio and fungal:bacterial ratios were calculated from control and treated soils where inhibition was measured as CO₂ respiration reduction with biocides. We were able to minimize non-target inhibition by the antibiotics to <5% and thus calculate reliable fungal:bacterial ratios using captan to inhibit fungi in all four soils, and bronopol to inhibit bacteria in three of the four soils. The most successful bactericide in the restored prairie was oxytetracycline-HCl. Our results demonstrate that application of novel antibiotics is not uniformly successful in soils of different origin and that the SI technique requires more than just optimization of antibiotic concentration; it also requires optimization of antibiotic selection.