Schmidt SK, Reed SC, Nemergut DR, Grandy AS, Cleveland CC, Weintraub MN, Hill AW, Costellow EK, Meyer AF, Neff JC, Martin AM. 2008. The earliest stages of ecosystem succession in high-elevation (5000 metres above sea level), recently deglaciated soils. Proceedings of the Royal Society of London, Series B 275: 2793-2802.
These authors describe the microbial community and soil parameters of a chronosequence at the foreground of a receding glacier high in the Peruvian Andes. From a combination of aerial photography and previous work at this site, a series of sites of soils of increasing ages from zero to 79 years old was established. No surface plants, even lichens, are present on any of these new soils, and soil nutrient levels (carbon, nitrogen) are very low; the only organisms present are microorganisms.
Two previous hypotheses had been proposed to explain the dynamics of very early primary succession on new soil. Organic matter has been observed to accumulate slowly in new soils; the source of this material is either aeolian deposits (i.e. wind-borne plant detritus and pollen) or in-situ fixation of CO2 and N2. These are not mutually exclusive hypotheses, but the relative contributions of each are explored in this study.
The methods used here cover an extensive list of soil parameters. Three sets of soil samples were collected: for microbiological analysis, N-fixation measurement, and all other chemical analyses. The other chemical analyses include photosynthetic pigment extraction, soil total and mineral nitrogen, pyrolysis for identifying sources of carbon compounds (i.e. microbial-autotroph, microbial-heterotroph, plant), enzyme assays for common and informative microbial enzymes, and soil stability analysis of the resistance of these new soils to erosion forces such as water runoff.
These authors focused on the cyanobacterial fraction of the microbial community in this study; some details of other components of the biota are described in an earlier paper, Nemergut et al. (2007). Cyanobacteria are autotrophs also capable of fixing atmospheric nitrogen, thus they are ideal primary colonizers of new soil as they require little more than a source of moisture and air. Analysis of the community included the use of the P-test (Martin 2002); note that as in Nemergut et al. (2007), he is one of the authors of this study. The analytical approach is very similar to that employed in the earlier study, with a comparison of discovered sequences to published sequences from around the world. In this study, cyanobacterial sequences from zero and 4-year-old soils were similar to sequences from an extremely broad sample of habitats, including Antarctic lake ice, marine subseafloor sediments, urban aerosols, forest soils, and oil-polluted soils.
The soil chronosequence showed a clear pattern of stages of primary succession at every level of analysis. The soil microbial community became both more abundant and more diverse through time, soil nutrients increased, the chemical environment included increasing amounts and diversity of complex organic molecules, key enzyme pathways became established, and soil stability increased as soils aged. N-fixation showed a peak, with increasing N-fixation activity from the zero to 4-year-old soils (by two orders of magnitude), then declining by about half in the 79-year-old soils. This mirrors and precedes a widely-observed pattern in plant primary succession, in which nitrogen-fixing plants are among the first colonizers, but decline in abundance at later stages of succession. Enzyme and organic molecule patterns were consistent with a total absence of heterotrophs in the extremely young soils, increasing occurrence of organisms capable of decomposing plant matter in the 4-year-old soils, and a molecular ecology qualitatively similar to a mature plant-associated soil in the 79-year-old soil.
The list of procedures and level of detail of analysis in this paper is impressive. Many, though certainly not all, of these techniques will be models for my own work, especially in the summer of 2010. The molecular-diversity techniques pioneered by Martin (Martin 2002, Nemergut et al. 2007, this paper) as well as the techniques of analyzing soil pigments and soil nutrients are all very interesting.
Thursday, April 1, 2010
Wednesday, March 31, 2010
Sørensen et al. 2006
Sørensen LI, Holmstrup M, Maraldo K, Christensen S, Christensen B. 2006. Soil fauna communities and microbial respiration in high Arctic tundra soils at Zackenberg, Northeast Greenland. Polar Biology 29: 189-195.
These authors sampled soil animals from three sites at Zackenberg station, Greenland, over three days in mid-summer. Two of the sites were considered mesic heath, with a mix of Cassiope tetragona and other High Arctic species of plants, while the third site was dominated by Dryas spp. and was considered dry heath; snow melts from the dry heath up to 20 days earlier than from the mesic heaths. Soil samples ranging down to about 6cm depth were collected, stored at 5ºC for up to two weeks, and analyzed by a range of methods in the laboratories in Europe.
Different groups of soil animals were extracted by varying methods. Soil microarthropods, a diverse group dominated by Collebola and Acari, were extracted by modified MacFadyen funnels into Benzoic acid. Enchytraeids and dipteran larvae were extracted in Baermann wet funnels with heating of the samples, into tap water. Protozoa were washed from soils in water and grown on media plates in the dark at 10ºC. Nematodes were collected by the Blender-Cotton wool method of Schouten and Arp (1991). Soil microbial respiration was measured in serum bottles, with the CO2 concentration in the headspace measured at zero, 5 and 25 hours, with a fully factorial design of nutrient amendments of C, N, and P. Soil pH and soil organic matter content, but not moisture content or other nutrient concentrations were determined using methods not clearly described, though presumably these procedures were similar to standard methods.
Once abundance and biomass data was collected, comparisons between plots were made using multivariate analysis and a software package named PRIMER 5.0. My understanding is the species counts were (log+1) transformed to reduce the influence of very abundant species, then analyzed using an approach similar to Principal Components Analysis. The result of this analysis was a clear difference between the dry heath and the two mesic heaths, while the two mesic heaths were not different from each other in parameter-space. A Bray-Curtis similarity matrix was also involved, though I’m not certain I understand how.
Different taxonomic groups were identified to different taxonomic levels; 19 species of Collembola and 7 species of Enchytraeids were found, for example, but Acari were identified to suborder (Cryptostigmata (oribatids), Prostigmata, Mesostigmata) and nematodes and protozoans were counted at those high taxonomic levels. While the two mesic heath sites were only marginally significantly different from each other, there was a clear increase in abundances in the dry heath site. For collembola at least, the dry heath site was also dominated by two highly abundant species, which differed from the majority of species in the mesic sites by being unpigmented and associated with sub-surface, rather than soil-surface, regions in the soil. The higher abundance of probably bacteria-eating nematodes at the dry heath strongly suggests higher turnover of microorganisms as well as generally higher biological activity from the higher populations of most soil animals.
These authors suggest higher organic matter decomposition rates at the dry heath, which seems reasonable given the higher animal populations there. However, their attribution of higher soil pH there to higher respiration levels seems like more of a stretch, absent supporting mineralogical and soil-nutrient data.
This paper provides an excellent example of the data that can be collected and analyzed from a brief but intensive study of soil invertebrates at a High Arctic site. In addition, meaningful information about differences in biodiversity between locations can be derived from studies of organisms not identified to fine taxonomic levels.
These authors sampled soil animals from three sites at Zackenberg station, Greenland, over three days in mid-summer. Two of the sites were considered mesic heath, with a mix of Cassiope tetragona and other High Arctic species of plants, while the third site was dominated by Dryas spp. and was considered dry heath; snow melts from the dry heath up to 20 days earlier than from the mesic heaths. Soil samples ranging down to about 6cm depth were collected, stored at 5ºC for up to two weeks, and analyzed by a range of methods in the laboratories in Europe.
Different groups of soil animals were extracted by varying methods. Soil microarthropods, a diverse group dominated by Collebola and Acari, were extracted by modified MacFadyen funnels into Benzoic acid. Enchytraeids and dipteran larvae were extracted in Baermann wet funnels with heating of the samples, into tap water. Protozoa were washed from soils in water and grown on media plates in the dark at 10ºC. Nematodes were collected by the Blender-Cotton wool method of Schouten and Arp (1991). Soil microbial respiration was measured in serum bottles, with the CO2 concentration in the headspace measured at zero, 5 and 25 hours, with a fully factorial design of nutrient amendments of C, N, and P. Soil pH and soil organic matter content, but not moisture content or other nutrient concentrations were determined using methods not clearly described, though presumably these procedures were similar to standard methods.
Once abundance and biomass data was collected, comparisons between plots were made using multivariate analysis and a software package named PRIMER 5.0. My understanding is the species counts were (log+1) transformed to reduce the influence of very abundant species, then analyzed using an approach similar to Principal Components Analysis. The result of this analysis was a clear difference between the dry heath and the two mesic heaths, while the two mesic heaths were not different from each other in parameter-space. A Bray-Curtis similarity matrix was also involved, though I’m not certain I understand how.
Different taxonomic groups were identified to different taxonomic levels; 19 species of Collembola and 7 species of Enchytraeids were found, for example, but Acari were identified to suborder (Cryptostigmata (oribatids), Prostigmata, Mesostigmata) and nematodes and protozoans were counted at those high taxonomic levels. While the two mesic heath sites were only marginally significantly different from each other, there was a clear increase in abundances in the dry heath site. For collembola at least, the dry heath site was also dominated by two highly abundant species, which differed from the majority of species in the mesic sites by being unpigmented and associated with sub-surface, rather than soil-surface, regions in the soil. The higher abundance of probably bacteria-eating nematodes at the dry heath strongly suggests higher turnover of microorganisms as well as generally higher biological activity from the higher populations of most soil animals.
These authors suggest higher organic matter decomposition rates at the dry heath, which seems reasonable given the higher animal populations there. However, their attribution of higher soil pH there to higher respiration levels seems like more of a stretch, absent supporting mineralogical and soil-nutrient data.
This paper provides an excellent example of the data that can be collected and analyzed from a brief but intensive study of soil invertebrates at a High Arctic site. In addition, meaningful information about differences in biodiversity between locations can be derived from studies of organisms not identified to fine taxonomic levels.
O'Neill et al. 2010
O’Neill KP, Godwin HW, Jiménez-Esquilín AE, Battigelli JP. 2010. Reducing the dimensionality of soil microinvertebrate community datasets using Indicator Species Analysis: Implications for ecosystem monitoring and soil management. Soil Biology & Biochemistry 42: 145-154.
These authors used a dataset of soil microarthropods to evaluate a method for identifying indicator species for ecosystem monitoring. The method centres on the Indicator Value (IV) of a species, a number that integrates the degree of uniqueness to a place of a species and the abundance of that species within a given habitat. A high IV value indicates both high information content and a high probability of being sampled. The IV is apparently robust to differences in site number and species absolute abundances, and provides a single value for evaluating observed or expected changes in an ecosystem. Indicator species, furthermore, integrate habitat conditions over their lifespans, in contrast to measures of chemical and physical parameters that capture a snapshot of an ecosystem.
The basic evaluation approach here was to identify indicator species along a clear environmental gradient from meadow to forest in West Virginia. The habitat was divided into three zones, with an edge patch between the open meadows and closed-canopy forest. Near-surface soil cores were collected from each zone every month from April 2004 to April 2005 (n = 180), using the top of the mineral soil as the reference depth. Microarthropods were extracted in a modified Macfadyen funnel with a strong and increasing temperature gradient, into 70% ethanol.
Diversity measures, including Simpson’s and Shannon indices, were based on counts of individuals identified to family level (suborder for Acari). Differences between sites were analyzed by 2-way repeated measures ANOVA and Principle Components Analysis, after rare taxa (those that occurred in less than 10% of samples) were removed; rare taxa are extremely unlikely to be identified as indicator species.
Calculating IV for each taxon, regardless of the taxonomic resolution, provides large advantages in labour time and taxonomic expertise, as many microfauna are very difficult to identify to genus or species. These authors state that enumeration of a single sample required more than 1 hour of a trained taxonomist’s time. In studies such as this one, there are further advantages of IV associated with its robustness in the face of many zero measurements (i.e. taxa absent from samples) and the general messiness of these kinds of datasets. However, the ISA approach is intended for 2-stage studies, where an intensive initial survey identifies indicator species (taxa), and later long-term monitoring ignores other species. For studies specificially designed to address biodiversity, such as my own, excluding rare taxa would not be beneficial, and there may be no easy escape from time-consuming morphotaxa sorting.
I have spoken with Dr. Battigelli, the trained taxonomist in this study. He has indicated that while this IV-based approach may not be appropriate for my own work, it nonetheless demonstrates the types of analyses that can be conducted with soil invertebrates identified to middle taxonomic levels. He has assured me I could probably be trained to identify Collembola to Family and Acari to Suborder in a matter of a few days, and he would be interested in futher studies of collected soil invertebrates based on interesting patterns that emerge at these taxonomic levels.
These authors used a dataset of soil microarthropods to evaluate a method for identifying indicator species for ecosystem monitoring. The method centres on the Indicator Value (IV) of a species, a number that integrates the degree of uniqueness to a place of a species and the abundance of that species within a given habitat. A high IV value indicates both high information content and a high probability of being sampled. The IV is apparently robust to differences in site number and species absolute abundances, and provides a single value for evaluating observed or expected changes in an ecosystem. Indicator species, furthermore, integrate habitat conditions over their lifespans, in contrast to measures of chemical and physical parameters that capture a snapshot of an ecosystem.
The basic evaluation approach here was to identify indicator species along a clear environmental gradient from meadow to forest in West Virginia. The habitat was divided into three zones, with an edge patch between the open meadows and closed-canopy forest. Near-surface soil cores were collected from each zone every month from April 2004 to April 2005 (n = 180), using the top of the mineral soil as the reference depth. Microarthropods were extracted in a modified Macfadyen funnel with a strong and increasing temperature gradient, into 70% ethanol.
Diversity measures, including Simpson’s and Shannon indices, were based on counts of individuals identified to family level (suborder for Acari). Differences between sites were analyzed by 2-way repeated measures ANOVA and Principle Components Analysis, after rare taxa (those that occurred in less than 10% of samples) were removed; rare taxa are extremely unlikely to be identified as indicator species.
Calculating IV for each taxon, regardless of the taxonomic resolution, provides large advantages in labour time and taxonomic expertise, as many microfauna are very difficult to identify to genus or species. These authors state that enumeration of a single sample required more than 1 hour of a trained taxonomist’s time. In studies such as this one, there are further advantages of IV associated with its robustness in the face of many zero measurements (i.e. taxa absent from samples) and the general messiness of these kinds of datasets. However, the ISA approach is intended for 2-stage studies, where an intensive initial survey identifies indicator species (taxa), and later long-term monitoring ignores other species. For studies specificially designed to address biodiversity, such as my own, excluding rare taxa would not be beneficial, and there may be no easy escape from time-consuming morphotaxa sorting.
I have spoken with Dr. Battigelli, the trained taxonomist in this study. He has indicated that while this IV-based approach may not be appropriate for my own work, it nonetheless demonstrates the types of analyses that can be conducted with soil invertebrates identified to middle taxonomic levels. He has assured me I could probably be trained to identify Collembola to Family and Acari to Suborder in a matter of a few days, and he would be interested in futher studies of collected soil invertebrates based on interesting patterns that emerge at these taxonomic levels.
Thursday, March 25, 2010
Harding et al. 2001
Harding RJ, Gryning S-E, Halldin S, Lloyd CR. 2001. Progress in understanding of land surface/atmosphere exchanges at high latitudes. Theoretical and Applied Climatology 70: 5-18.
These authors review and discuss the implications of studies based in two international projects in northern Europe. WINTEX was a large study examining the effects of snow cover and long nights in winter on high-latitude ground-atmosphere exchange processes, while LAPP was an independent but complementary study examining most of the same processes in a range of high latitude sites during spring and summer.
Snow cover plays a major role in Arctic exchange processes. The high albedo of snow reflects much of the incident solar radiation, and insulates the frozen ground below, prolonging the period of snow cover to upwards of 9 months in the year in many places. Where vegetation is tall, such as in the boreal forest, the low solar angle reduces the effective net albedo of the landscape, allowing sunlight to warm the dark trees and speed springtime melting. This study mentions the importance of snow-surface aerodynamics, though it appears there is little solid information on this complex topic.
Snow melt is the major hydrological event of the year in much of the Arctic. The combination of frozen soils, very low evaporation rates, and often flat terrain means much of the Arctic is very wet or saturated while annual precipitation rates are consistent with arid or semi-arid conditions. These areas are the classic tundra systems, with abundant shallow lakes and ponds and very wet high-organic soils.
Differences in snow-surface dynamics and the timing of snowmelt create an extremely heterogeneous landscape, particularly in the vicinity of the northern treeline. There are often very large temperature and air-flow differences between patches of trees and adjacent lakes or clearings, which greatly complicate attempts to model the carbon dioxide emissions (for example) of such areas. Much of this paper is a series of evaluations of some of the models that have been applied to this region. In general, more sophisticated models that can take some of the extreme variability into account perform better than models that cannot account for differences in snow depth or insulating properties. However, this paper makes it clear that current modelling efforts still leave much to be desired in terms of predicting Arctic heat budgets and biological responses.
Water storage is also very difficult to model, and has large and variable impacts on other parts of the system. There appears to be large and unpredictable year-to-year variation in water storage and transport at the scale of catchments and basins, and the importance of soil water in controlling biological processes such as the decomposition of organic matter is large. Runoff matters, even on very gentle slopes.
This paper provides a useful overview of large-scale processes and attempts to understand these processes in the Arctic.
These authors review and discuss the implications of studies based in two international projects in northern Europe. WINTEX was a large study examining the effects of snow cover and long nights in winter on high-latitude ground-atmosphere exchange processes, while LAPP was an independent but complementary study examining most of the same processes in a range of high latitude sites during spring and summer.
Snow cover plays a major role in Arctic exchange processes. The high albedo of snow reflects much of the incident solar radiation, and insulates the frozen ground below, prolonging the period of snow cover to upwards of 9 months in the year in many places. Where vegetation is tall, such as in the boreal forest, the low solar angle reduces the effective net albedo of the landscape, allowing sunlight to warm the dark trees and speed springtime melting. This study mentions the importance of snow-surface aerodynamics, though it appears there is little solid information on this complex topic.
Snow melt is the major hydrological event of the year in much of the Arctic. The combination of frozen soils, very low evaporation rates, and often flat terrain means much of the Arctic is very wet or saturated while annual precipitation rates are consistent with arid or semi-arid conditions. These areas are the classic tundra systems, with abundant shallow lakes and ponds and very wet high-organic soils.
Differences in snow-surface dynamics and the timing of snowmelt create an extremely heterogeneous landscape, particularly in the vicinity of the northern treeline. There are often very large temperature and air-flow differences between patches of trees and adjacent lakes or clearings, which greatly complicate attempts to model the carbon dioxide emissions (for example) of such areas. Much of this paper is a series of evaluations of some of the models that have been applied to this region. In general, more sophisticated models that can take some of the extreme variability into account perform better than models that cannot account for differences in snow depth or insulating properties. However, this paper makes it clear that current modelling efforts still leave much to be desired in terms of predicting Arctic heat budgets and biological responses.
Water storage is also very difficult to model, and has large and variable impacts on other parts of the system. There appears to be large and unpredictable year-to-year variation in water storage and transport at the scale of catchments and basins, and the importance of soil water in controlling biological processes such as the decomposition of organic matter is large. Runoff matters, even on very gentle slopes.
This paper provides a useful overview of large-scale processes and attempts to understand these processes in the Arctic.
Thursday, March 11, 2010
Martin and Rygiewicz 2005
Martin KJ, Rygiewicz PT. 2005. Fungal-specific PCR primers developed for analysis of the ITS region of environmental DNA extracts. BMC Microbiology 5:28.
These authors designed new primers for PCR and related molecular biology investigations of soil fungi, especially mycorrhizae. These are very diverse organisms, and, because of the commonalities between fungal and plant DNA, studies of fungal samples closely entwined with plant tissue as in root-associated mycorrhizae can be very complex. The new primers were designed to produce a range of PCR products suitable for techniques such as qPCR, Length-Heterogeneity PCR (LH-PCR) and T-RFLP analyses.
The primers as a suite were designed around a nested approach, with new outer primers amplifying a long DNA sequence of approximately 1000bp, and later primer pairs amplifying regions within that long sequence. Most inner primer pairs generate products of approximately 500bp length.
These authors also used a different DNA extraction method, based on xanthogenate and Tween (X/T) that involves little to no tissue grinding, compared to the standard method based on CTAB. The X/T method preferentially extracts fungal DNA from cells on the outside of particles, for example fungal cells not penetrating plant roots. This reduces the amount of plant DNA and associated plant-derived compounds in resulting extracts. Combining the two techniques may allow for some interesting studies of fungal micro-ecology.
This paper’s novel primers should be more specific and more useful to my own qPCR studies of Arctic soil microbes, including soil fungi. The methods section detailing some of the decisions made and considerations involved in primer design will also be useful.
These authors designed new primers for PCR and related molecular biology investigations of soil fungi, especially mycorrhizae. These are very diverse organisms, and, because of the commonalities between fungal and plant DNA, studies of fungal samples closely entwined with plant tissue as in root-associated mycorrhizae can be very complex. The new primers were designed to produce a range of PCR products suitable for techniques such as qPCR, Length-Heterogeneity PCR (LH-PCR) and T-RFLP analyses.
The primers as a suite were designed around a nested approach, with new outer primers amplifying a long DNA sequence of approximately 1000bp, and later primer pairs amplifying regions within that long sequence. Most inner primer pairs generate products of approximately 500bp length.
These authors also used a different DNA extraction method, based on xanthogenate and Tween (X/T) that involves little to no tissue grinding, compared to the standard method based on CTAB. The X/T method preferentially extracts fungal DNA from cells on the outside of particles, for example fungal cells not penetrating plant roots. This reduces the amount of plant DNA and associated plant-derived compounds in resulting extracts. Combining the two techniques may allow for some interesting studies of fungal micro-ecology.
This paper’s novel primers should be more specific and more useful to my own qPCR studies of Arctic soil microbes, including soil fungi. The methods section detailing some of the decisions made and considerations involved in primer design will also be useful.
Wednesday, February 24, 2010
Philippot et al. 2002
Philippot L, Piutti S, Martin-Laurent F, Hallet S, Germon JC. 2002. Molecular analysis of the nitrate-reducing community from unplanted and maize-planted soils. Applied and Environmental Microbiology 68: 6121-6128.
These authors applied molecular techniques including PCR, RFLP, and sequencing to the study of soil bacteria relevant to crops. Dissimilatory nitrate reduction, the process that converts NO3- to NO2-, is widespread in prokaryotes, with the activity described in alpha, beta, and gamma Proteobacteria, gram-positive bacteria, and some archaea. There are two described enzymes that catalyze the reaction and provide energy to the organism; these authors focused on the membrane-bound protein, specifically one subunit that includes a distinctive set of components. Their approach was to design primers for a well-conserved region of the gene narG that amplify a 650bp region, and then subject the PCR product to cloning, RFLP analysis, and sequencing.
Community structure and diversity was compared between pots planted with maize versus unplanted controls. Maize (Zea mays) is a plant that facilitates gas diffusion in its roots under oxygen-stress soil conditions; this creates an aerobic region in the rhizosphere distinct from anaerobic conditions further from roots. While diversity, as measured by standard indices, did not differ between planted and unplanted soils, the structure of the communities did change, with numerous RFLP phylotypes found in only one or the other treatment. This suggests a role of rhizosphere conditions, likely involving both oxygen and root exudates, in selecting for particular groups of microorganisms.
Nitrate reduction occurs primarily or possibly only under aerobic conditions. The microbial cell gains energy from dissimilatory reduction of nitrate, and if it occurs in the rhizosphere, the plant may gain a readily-accessible form of nitrogen in the form of nitrite. Denitrification, the process that shuttles nitrogen atoms from nitrite to gaseous forms such as N2O or N2, can occur under a range of oxygen conditions, including aerobic, thus denitrifiers in the rhizosphere may compete with plant roots for nitrite. The fate of nitrite produced by dissimilatory nitrate reduction can also be to ammonium, though this appears to be rare in soil and more common in vertebrate guts and digested sludge, two environments typically lacking in oxygen.
This paper provides some molecular tools for my own studies of nitrogen dynamics in soils, especially the sequences of the degenerate primers. In addition, it provides some clarification of parts of the remarkably complex soil-nitrogen cycle.
These authors applied molecular techniques including PCR, RFLP, and sequencing to the study of soil bacteria relevant to crops. Dissimilatory nitrate reduction, the process that converts NO3- to NO2-, is widespread in prokaryotes, with the activity described in alpha, beta, and gamma Proteobacteria, gram-positive bacteria, and some archaea. There are two described enzymes that catalyze the reaction and provide energy to the organism; these authors focused on the membrane-bound protein, specifically one subunit that includes a distinctive set of components. Their approach was to design primers for a well-conserved region of the gene narG that amplify a 650bp region, and then subject the PCR product to cloning, RFLP analysis, and sequencing.
Community structure and diversity was compared between pots planted with maize versus unplanted controls. Maize (Zea mays) is a plant that facilitates gas diffusion in its roots under oxygen-stress soil conditions; this creates an aerobic region in the rhizosphere distinct from anaerobic conditions further from roots. While diversity, as measured by standard indices, did not differ between planted and unplanted soils, the structure of the communities did change, with numerous RFLP phylotypes found in only one or the other treatment. This suggests a role of rhizosphere conditions, likely involving both oxygen and root exudates, in selecting for particular groups of microorganisms.
Nitrate reduction occurs primarily or possibly only under aerobic conditions. The microbial cell gains energy from dissimilatory reduction of nitrate, and if it occurs in the rhizosphere, the plant may gain a readily-accessible form of nitrogen in the form of nitrite. Denitrification, the process that shuttles nitrogen atoms from nitrite to gaseous forms such as N2O or N2, can occur under a range of oxygen conditions, including aerobic, thus denitrifiers in the rhizosphere may compete with plant roots for nitrite. The fate of nitrite produced by dissimilatory nitrate reduction can also be to ammonium, though this appears to be rare in soil and more common in vertebrate guts and digested sludge, two environments typically lacking in oxygen.
This paper provides some molecular tools for my own studies of nitrogen dynamics in soils, especially the sequences of the degenerate primers. In addition, it provides some clarification of parts of the remarkably complex soil-nitrogen cycle.
Siciliano et al. 2007
Siciliano SD, Ma W, Powell S. 2007. Evaluation of quantitative polymerase chain reaction to assess nosZ gene prevalence in mixed microbial communities. Canadian Journal of Microbiology 53: 636-642.
These authors examined the usefulness of qPCR in studying populations of soil bacteria, especially denitrifiers using the gene nosZ that codes for nitrous oxide reductase. This enzyme catalyzes the final reaction in the process of denitrification, converting N2O to N2. Normally, it is expressed only in severely anaerobic conditions, as it allows the use of N2O as the terminal electron acceptor during metabolism.
There are a number of factors that control the efficiency of PCR in quantitative PCR applications. The efficiency is a major component of the calculations that allow qPCR to estimate gene copy numbers in samples and thus to be used to examine population dynamics of non-culturable microorganisms from environmental samples. Of particular importance is consistency of efficiency between the amplification of the standard DNA template and the amplification of all templates in the unknown samples. Variation between the standard and the unknowns can lead to severe under- or over-estimation of target populations, while variation in efficiency between different templates within the unknown samples can lead to misestimations of relative proportions of organisms.
These authors evaluated the efficiency of qPCR in a range of experimental templates, and in a range of combinations simulating mixed populations. Little variance in efficiency was found, and this variance was not associated with genetic distance from a reference organism. The experimental design did not allow a direct examination of the influence of the geographical differences in the sources of the test sequences (Arctic, temperate-grassland, Antarctic), but this lack of association with the reference organism does indicate low or no variation among PCR efficiencies associated with some other variable.
The influence of varying PCR efficiencies among templates within a sample becomes less severe as the number of different templates rises. In a typical soil sample with perhaps 1000 different templates, no one template can utterly dominate amplification by outcompeting for primers, thus the resulting mix of amplicons at the end of 40 rounds of PCR will most likely be representative of the population mixture in the environment.
This paper is of obvious high utility to my own work, not least because the individual machine used to perform qPCR is the same individual machine that I will be using. For this and other reasons, this paper was suggested to me, repeatedly. Future reference to this paper, when I am developing my methods and when I am writing up the next paper or two, seems likely.
These authors examined the usefulness of qPCR in studying populations of soil bacteria, especially denitrifiers using the gene nosZ that codes for nitrous oxide reductase. This enzyme catalyzes the final reaction in the process of denitrification, converting N2O to N2. Normally, it is expressed only in severely anaerobic conditions, as it allows the use of N2O as the terminal electron acceptor during metabolism.
There are a number of factors that control the efficiency of PCR in quantitative PCR applications. The efficiency is a major component of the calculations that allow qPCR to estimate gene copy numbers in samples and thus to be used to examine population dynamics of non-culturable microorganisms from environmental samples. Of particular importance is consistency of efficiency between the amplification of the standard DNA template and the amplification of all templates in the unknown samples. Variation between the standard and the unknowns can lead to severe under- or over-estimation of target populations, while variation in efficiency between different templates within the unknown samples can lead to misestimations of relative proportions of organisms.
These authors evaluated the efficiency of qPCR in a range of experimental templates, and in a range of combinations simulating mixed populations. Little variance in efficiency was found, and this variance was not associated with genetic distance from a reference organism. The experimental design did not allow a direct examination of the influence of the geographical differences in the sources of the test sequences (Arctic, temperate-grassland, Antarctic), but this lack of association with the reference organism does indicate low or no variation among PCR efficiencies associated with some other variable.
The influence of varying PCR efficiencies among templates within a sample becomes less severe as the number of different templates rises. In a typical soil sample with perhaps 1000 different templates, no one template can utterly dominate amplification by outcompeting for primers, thus the resulting mix of amplicons at the end of 40 rounds of PCR will most likely be representative of the population mixture in the environment.
This paper is of obvious high utility to my own work, not least because the individual machine used to perform qPCR is the same individual machine that I will be using. For this and other reasons, this paper was suggested to me, repeatedly. Future reference to this paper, when I am developing my methods and when I am writing up the next paper or two, seems likely.
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