Catherine E. Dandie

Changes in denitrifier abundance, denitrification gene mRNA levels, nitrous oxide emissions and denitrification in anoxic soil microcosms amended with glucose and plant residues.

ABSTRACT In agricultural cropping systems, crop residues are sources of organic carbon (C), an important factor influencing denitrification. The effects of red clover, soybean, and barley plant residues and of glucose on denitrifier abundance, denitrification gene mRNA levels, nitrous oxide (N2O) emissions, and denitrification rates were quantified in anoxic soil microcosms for 72 h. nosZ gene abundances and mRNA levels significantly increased in response to all organic carbon treatments over time. In contrast, the abundance and mRNA levels of Pseudomonas mandelii and closely related species (nirSP) increased only in glucose-amended soil: the nirSP guild abundance increased 5-fold over the 72-h incubation period (P < 0.001), while the mRNA level significantly increased more than 15-fold at 12 h (P < 0.001) and then subsequently decreased. The nosZ gene abundance was greater in plant residue-amended soil than in glucose-amended soil. Although plant residue carbon-to-nitrogen (C:N) ratios varied from 15:1 to 30:1, nosZ gene and mRNA levels were not significantly different among plant residue treatments, with an average of 3.5 × 107 gene copies and 6.9 × 107 transcripts g−1 dry soil. Cumulative N2O emissions and denitrification rates increased over 72 h in both glucose- and plant-tissue-C-treated soil. The nirSP and nosZ communities responded differently to glucose and plant residue amendments. However, the targeted denitrifier communities responded similarly to the different plant residues under the conditions tested despite changes in the quality of organic C and different C:N ratios.

Changes in bacterial denitrifier community abundance over time in an agricultural field and their relationship with denitrification activity

ABSTRACT This study measured total bacterial and denitrifier community abundances over time in an agricultural soil cropped to potatoes (Solanum tuberosum L.) by using quantitative PCR. Samples were collected on 10 dates from spring to autumn and from three spatial locations: in the potato “hill” between plants (H), close to the plant (Hp), and in the “furrow” (F). The denitrification rates, N2O emissions, and environmental parameters were also measured. Changes in denitrifier abundance over time and spatial location were small (1.7- to 2.7-fold for the nirK, nosZ, and cnorBB guilds), whereas the cnorBP community (Pseudomonas mandelii and closely related spp.) showed an ∼4.6-fold change. The seasonal patterns of denitrifier gene numbers varied with the specific community: lower nosZ gene numbers in April and May than in June and July, higher cnorBP gene numbers in May and June than in March and April and September and November, higher nirK gene numbers in early spring than in late autumn, and no change in cnorBB gene numbers. Gene numbers were higher for the Hp than the H location for the nosZ and nirK communities and for the cnorBP community on individual dates, presumably indicating an effect of the plant on denitrifier abundance. Higher cnorBP gene numbers for the H location than the F location and for nosZ and cnorBB on individual dates reflect the effect of spatial location on abundance. Denitrifier abundance changes were not related to any environmental parameter, although a weak relationship exists between cnorBP gene numbers, extractable organic carbon values, and temperature. Denitrification and N2O emissions were mostly regulated by inorganic nitrogen availability and water-filled pore space but were uncoupled from denitrifier community abundances measured in this system.

Abundance, diversity and functional gene expression of denitrifier communities in adjacent riparian and agricultural zones

Lands under riparian and agricultural management differ in soil properties, water content, plant species and nutrient content and are therefore expected to influence denitrifier communities, denitrification and nitrous oxide (N(2) O) emissions. Denitrifier community abundance, denitrifier community structure, denitrification gene expression and activity were quantified on three dates in a maize field and adjacent riparian zone. N(2) O emissions were greater in the agricultural zone, whereas complete denitrification to N(2) was greater in the riparian zone. In general, the targeted denitrifier community abundance did not change between agricultural and riparian zones. However, nosZ gene expression was greater in the riparian zone than the agricultural zone. The community structure of nirS-gene-bearing denitrifiers differed in June only, whereas the nirK-gene-bearing community structure differed significantly between the riparian and the agricultural zones at all dates. The nirK-gene-bearing community structure was correlated with soil pH, while no significant correlations were found between nirS-gene-bearing community structure and soil environmental variables or N(2) O emissions, denitrification or denitrifier enzyme activity. The results suggested for the nirK and nirS-gene-bearing communities different factors control abundance vs. community structure. The nirK-gene-bearing community structure was also more responsive than the nirS-gene-bearing community structure to change between the two ecosystems.

Nitric Oxide Reductase-Targeted Real-Time PCR Quantification of Denitrifier Populations in Soil

ABSTRACT The quantification of denitrifying bacteria is a component in the further understanding of denitrification processes in the environment. Real-time PCR primers were designed to target two segments of the denitrifier population (cnorBP [Pseudomonas mandelii and closely related strains] and cnorBB [Bosea, Bradyrhizobium, and Ensifer spp.]) in agricultural soils based on functional cnorB (nitric oxide reductase) gene sequences. Total population numbers were measured using 16S rRNA gene real-time PCR. Two soil microcosm experiments were conducted. Experiment 1 examined the response of the indigenous soil microbial population to the addition of 500 mg/kg glucose-C daily over 7 days in soil microcosms. Changes in the total population were correlated (r = 0.83) between 16S rRNA gene copy numbers and microbial biomass carbon estimates. Members of the cnorBP population of denitrifiers showed typical r-strategy by being able to increase their proportion in the total population from starting levels of <0.1% to around 2.4% after a daily addition of 500 mg/kg glucose-C. The cnorBB guild was not able to increase its relative percentage of the total population in response to the addition of glucose-C, instead increasing copy numbers only in proportion with the total population measured by 16S rRNA genes. Experiment 2 measured population dynamics in soil after the addition of various amounts of glucose-C (0 to 500 mg/kg) and incubation under denitrifying conditions. cnorBP populations increased proportionally with the amount of glucose-C added (from 0 to 500 mg/kg). In soil microcosms, denitrification rates, respiration, and cnorBP population densities increased significantly with increasing rates of glucose addition. cnorBB guild densities did not increase significantly under denitrifying conditions in response to increasing C additions.

Diversity of nirK denitrifying genes and transcripts in an agricultural soil.

ABSTRACT Environmental conditions can change dramatically over a crop season and among locations in an agricultural field and can increase denitrification and emissions of the potent greenhouse gas nitrous oxide. In a previous study, changes in the overall size of the denitrifier community in a potato crop field were relatively small and did not correlate with variations in environmental conditions or denitrification rates. However, denitrifying bacteria are taxonomically diverse, and different members of the community may respond differently to environmental changes. The objective of this research was to understand which portion of the nirK denitrifying community is active and contributes to denitrification under conditions in a potato crop field. Denaturing gradient gel electrophoresis (DGGE) of nirK genes in soil-extracted DNA showed changes in the composition of the nirK denitrifier community over the growing season and among spatial locations in the field. By contrast, the composition of the active nirK denitrifier community, as determined by DGGE analysis of nirK transcripts derived from soil-extracted mRNA, changed very little over time, although differences in the relative abundance of some specific transcripts were observed between locations. Our results indicate that the soil denitrifier populations bearing nirK genes are not all contributing to denitrification and that the denitrifying populations that are active are among the most abundant and ubiquitous nirK-bearing denitrifiers. Changes in the community composition of the total and active nirK denitrifiers were not strongly correlated with changes in environmental factors and denitrification activity.

Abundance, diversity and spatio-temporal dynamics of nirS gene-harbouring denitrifiers in a potato field over the course of a growth season

The abundance and diversity of nirS-harbouring bacteria were evaluated in a potato field during a growth season using culture-independent techniques. A total of 182 operational taxonomical units were identified and most had low homology to known nirS sequences, which suggested the discovery of new denitrifiers. The diversity was significantly higher in the furrow, followed by the hill and the near-plant region and was inversely proportional to the denitrification enzyme activity. In contrast, the abundance was not altered by soil locations but was significantly lower at the end of the growth season.