Claudia Goyer

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.

Effect of pH and temperature on denitrification gene expression and activity in Pseudomonas mandelii.

ABSTRACT Pseudomonas mandelii liquid cultures were studied to determine the effect of pH and temperature on denitrification gene expression, which was quantified by quantitative reverse transcription-PCR. Denitrification was measured by the accumulation of nitrous oxide (N2O) in the headspace in the presence of acetylene. Levels of gene expression of nirS and cnorB at pH 5 were 539-fold and 6,190-fold lower, respectively, than the levels of gene expression for cells grown at pH 6, 7, and 8 between 4 h and 8 h. Cumulative denitrification levels were 28 μmol, 63 μmol, and 22 μmol at pH 6, 7, and 8, respectively, at 8 h, whereas negligible denitrification was measured at pH 5. P. mandelii cells grown at 20°C and 30°C exhibited 9-fold and 94-fold increases in levels of cnorB expression between 0 h and 2 h, respectively, and an average 17-fold increase in levels of nirS gene expression. In contrast, induction of cnorB and nirS gene expression for P. mandelii cells grown at 10°C did not occur in the first 4 h. Levels of cumulative denitrification at 10 h were 6.6 μmol for P. mandelii cells grown at 10°C and 20°C and 30 μmol for cells grown at 30°C. Overall, levels of cnorB and nirS expression were relatively insensitive to pH values over the range of pH 6 to 8 but were substantially reduced at pH 5, whereas gene expression was sensitive to temperature, with induction and time to achieve maximum gene expression delayed as the temperature decreased from 30°C. Low pH and temperature negatively affected denitrification activity.

Environmental stresses induce the expression of putative glycine-rich insect cuticular protein genes in adult Leptinotarsa decemlineata (Say).

The deposition of cuticular proteins in insects usually occurs during the moulting process. Three putative glycine‐rich insect cuticular proteins, Ld‐GRP1 to 3, were identified and characterized from the Colorado potato beetle, Leptinotarsa decemlineata. The Ld‐GRPs contained conserved GXGX and/or GGXG sequence repeats. Ld‐GRP1 also contained a conserved AAPA/V motif commonly found in cuticular proteins. The transcripts of Ld‐GRP1 and Ld‐GRP2 were detected in the epidermal cell layer by in situ hybridization, making them putative insect cuticular proteins. The putative cuticular protein genes were highly induced by the insecticide azinphosmethyl (organophosphorous) 2–3 weeks after adult moulting. Putative cuticular protein gene expression level was higher in azinphosmethyl‐resistant beetles than in susceptible beetles. Furthermore, two of the putative cuticular protein genes were highly induced by dry environmental conditions. These results suggest that the insect might increase cuticular component deposition in the adult stage in response to environmental stresses. This ability may allow the insect to adapt to new or changing environments.

A phloem-sap feeder mixes phloem and xylem sap to regulate osmotic potential

Phloem-sap feeders (Hemiptera) occasionally consume the dilute sap of xylem, a behaviour that has previously been associated with replenishing water balance following dehydration. However, a recent study reported that non-dehydrated aphids ingested xylem sap. Here, we tested the hypothesis that the consumption of xylem sap, which has a low osmolality, is a general response to osmotic stresses other than dehydration. Alate aphids were subjected to different treatments and subsequently transferred onto a plant, where electrical penetration graph (EPG) was used to estimate durations of passive phloem sap consumption and active sucking of xylem sap. The proportion of time aphids fed on xylem sap (i.e., time spent feeding on xylem sap/total time spent feeding on phloem plus xylem sap) was used as a proxy of the solute concentration of the uptake. The proportion of time alate aphids fed on xylem sap increased: (1) with the time spent imbibing an artificial diet containing a solution of sucrose, which is highly concentrated in phloem sap and is mainly responsible for the high osmotic potential of phloem sap; (2) with the osmotic potential of the artificial diet, when osmotic potential excess was not related to sucrose concentration; and (3) when aphids were deprived of primary symbionts, a condition previously shown to lead to a higher haemolymph osmotic potential. All our results converge to support the hypothesis that xylem sap consumption contributes to the regulation of the osmotic potential in phloem-sap feeders.

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.

Potato Expressed Sequence Tag Generation and Analysis using Standard and Unique cDNA Libraries

To help develop an understanding of the genes that govern the developmental characteristics of the potato (Solanum tuberosum), as well as the genes associated with responses to specified pathogens and storage conditions, The Canadian Potato Genome Project (CPGP) carried out 5′ end sequencing of regular, normalized and full-length cDNA libraries of the Shepody potato cultivar, generating over 66,600 expressed sequence tags (ESTs). Libraries sequenced represented tuber developmental stages, pathogen-challenged tubers, as well as leaf, floral developmental stages, suspension cultured cells and roots. All libraries analysed to date have contributed unique sequences, with the normalized libraries high on the list. In addition, a low molecular weight library has enhanced the 3′ ends of our sequence assemblies. Using the combined assembly dataset, unique tuber developmental, cold storage and pathogen-challenged sequences have been identified. A comparison of the ESTs specific to the pathogen-challenged tuber and foliar libraries revealed minimal overlap between these libraries. Mixed assemblies using over 189,000 potato EST sequences from CPGP and The Institute for Genomics Research (TIGR) has revealed common sequences, as well as CPGP- and TIGR-unique sequences.

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.

Effects of temperatures near the freezing point on N2O emissions, denitrification and on the abundance and structure of nitrifying and denitrifying soil communities

Climate warming in temperate regions may lead to decreased soil temperatures over winter as a result of reduced snow cover. We examined the effects of temperatures near the freezing point on N(2)O emissions, denitrification, and on the abundance and structure of soil nitrifiers and denitrifiers. Soil microcosms supplemented with NO3 - and/or NO3 - plus red clover residues were incubated for 120 days at -4 °C, -1 °C, +2 °C or +5 °C. Among microcosms amended with residues, N(2)O emission and/or denitrification increased with increasing temperature on Days 2 and 14. Interestingly, N(2)O emission and/or denitrification after Day 14 were the greatest at -1 °C. Substantial N(2) O emissions were only observed on Day 2 at +2 °C and +5 °C, while at -1 °C, N(2)O emissions were consistently detected over the duration of the experiment. Abundances of ammonia oxidizing bacteria (AOB) and archaea (AOA), Nitrospira-like bacteria and nirK denitrifiers were the lowest in soils at -4 °C, while abundances of Nitrobacter-like bacteria and nirS denitrifiers did not vary among temperatures. Community structures of nirK and nirS denitrifiers and Nitrobacter-like bacteria shifted between below-zero and above-zero temperatures. Structure of AOA and AOB communities also changed but not systematically among frozen and unfrozen temperatures. Results indicated shifts in some nitrifier and denitrifier communities with freezing and a surprising stimulation of N(2)O emissions at -1 °C when NO3 - and C are present.