Chemical fumigation and biofumigation alter soil bacterial community diversity and composition.

Chemical fumigation and biofumigation are used to reduce soil-borne diseases in agricultural production systems; however, non-targeted soil microorganisms may also be affected. This study compared the effects of chemical fumigation, either used alone or combined with an organic amendment, and biofumigation on soil bacterial community diversity and composition under controlled conditions over 160 days. Treatments included: fumigation with chloropicrin (CP), fumigation with metam sodium used alone (MS) or combined with barley plant residues (MSBR); biofumigation with mustard plant residues; addition of barley plant residues; and untreated control. Biofumigation had a greater impact on bacterial diversity at early time points, transiently decreasing species evenness and yielding the most dissimilar ß-diversity after 3 days. MS fumigation did not affect bacterial diversity indices; however, MSBR transiently decreased species evenness after 8 days. CP-treated soil had decreased species evenness that did not recover over time and had the most dissimilar ß-diversity at the end of the incubation compared to all other treatments. This study demonstrated that CP fumigation had the greatest and most persistent impact on bacterial diversity, whereas MS fumigation and biofumigation led to transient decreases in bacterial diversity.

Effect of Biofumigation on Population Densities of Pratylenchus spp. and Verticillium spp. and Potato Yield in Eastern Canada.

Biofumigation has been proposed as an alternative to soil fumigation to manage soil-borne diseases including potato early dying disease complex (PED). This study examined the potential of using brown mustard (Mustard juncea) biofumigation to manage PED under rain-fed potato production in New Brunswick, Canada in two trials between 2017 and 2020 in comparison with chloropicrin fumigation and a conventional barley rotation. Biofumigation increased yield in one trial, but not in a second trial where the potato crop experienced severe drought, whereas chloropicrin fumigation increased yield in both trials. Biofumigation was effective in suppressing root-lesion nematode (RLN, Pratylenchus spp.) counts in both trials, but was ineffective in suppressing V. dahliae population density. Chloropicrin fumigation was effective in suppressing RLN counts and V. dahliae population density only in the hill where injected, but the effect was short-lived as the population density of V. dahliae in the hill increased to the level of the control in one potato growing season. Biofumigation may be an alternative to chloropicrin fumigation in managing PED, particularly in fields with high RLN population but relatively low Verticillium population density. However, neither biofumigation nor fumigation used alone may be sustainable in the short-term potato rotations commonly used in New Brunswick, and additional beneficial practices are required to sustain productivity in the long-term.

La biofumigación se ha propuesto como una alternativa a la fumigación del suelo para manejar las enfermedades transmitidas por el suelo, incluido el complejo de enfermedades de muerte prematura de la papa (PED). Este estudio examinó el potencial del uso de la biofumigación de mostaza marrón (Mustard juncea) para manejar la PED bajo la producción de papa de secano en New Brunswick, Canadá, en dos ensayos entre 2017 y 2020 en comparación con la fumigación con cloropicrina y una rotación de cebada convencional. La biofumigación aumentó el rendimiento en un ensayo, pero no en un segundo ensayo en el que el cultivo de papa experimentó una sequía severa, mientras que la fumigación con cloropicrina aumentó el rendimiento en ambos ensayos. La biofumigación fue efectiva para suprimir los conteos del nematodo lesionador de la raíz (RLN, Pratylenchus spp.) en ambos ensayos, pero fue ineficaz para suprimir la densidad de población de V. dahliae. La fumigación con cloropicrina fue efectiva para suprimir los conteos de RLN y la densidad de población de V. dahliae solo en el lomo del surco donde se inyectó, pero el efecto fue de corta duración ya que la densidad de población de V. dahliae en el surco aumentó al nivel del testigo en un ciclo de cultivo de papa. La biofumigación puede ser una alternativa a la fumigación con cloropicrina en el manejo de la PED, particularmente en campos con alta población de RLN pero densidad de población de Verticillium relativamente baja. Sin embargo, ni la biofumigación ni la fumigación utilizadas por sí solas pueden ser sustentables en las rotaciones de papa a corto plazo comúnmente utilizadas en New Brunswick, y se requieren prácticas benéficas adicionales para mantener la productividad a largo plazo.

Seasonal changes in the abundance and activity of bacterial and fungal denitrifying communities associated with different compost amendments.

Composts can be efficient organic amendments in potato culture as they can supply carbon and nutrients to the soil. However, more information is required on the effects of composts on denitrification and nitrous oxide emissions (N2O) and emission-producing denitrifying communities. The effects of three compost amendments (municipal source separated organic waste compost (SSOC), forestry waste mixed with poultry manure compost (FPMC), and forestry residues compost (FRC)) on fungal and bacterial denitrifying communities and activity was examined in an agricultural field cropped to potatoes during the fall, spring, and summer seasons. The denitrification enzyme activity (DEA), N2O emissions, and respiration were measured in parallel. N2O emission rates were greater in FRC-amended soils in the fall and summer, whereas soil respiration was highest in the SSOC-amended soil in the fall. A large number of nirK denitrifying fungal transcripts were detected in the fall, coinciding with compost application, while the greatest nirK bacterial transcripts were measured in the summer when plants were actively growing. Denitrifying community and transcript levels were poor predictors of DEA, N2O emissions, or respiration rates in compost-amended soil. Overall, the sampling date was driving the population and activity levels of the three denitrifying communities under study.

Linking changes in snow cover with microbial nitrogen cycling functional gene abundance and expression in agricultural soil.

In eastern Canada, climate change-related warming and increased precipitation may alter winter snow cover, with potential consequences for soil conditions, nitrogen (N) cycling, and microbes. We conducted a 2-year field study aimed at determining the influence of snow removal, snow accumulation, and ambient snow in a potato-barley crop system on the abundance and expression of denitrifier (nirS, nirK, nosZ) and nitrifier (ammonium oxidizing archaeal (AOA) and bacterial (AOB) amoA) genes. Denitrifier and nitrifier abundance and expression results were compared to N2O production, soil atmosphere accumulation, and surface fluxes. In the first winter, nirK abundance was lowest while AOB abundance was greatest in snow accumulation treatments. In the second winter, greatest abundances were observed in the ambient snow treatment, which had greatest N2O accumulation and spring thaw fluxes, suggesting a link between microbial populations and biogeochemical functioning. Treatment effects on gene expression were limited, but greatest AOA, AOB, and nosZ expression was measured near 0°C and above 15°C, indicating that activity was promoted by freeze-thaw conditions and at summer temperatures. Overall, effects of changing snow depth on denitrifier and nitrifier abundance were not solely due to change in soil temperature, but also to soil moisture and/or interactions between these parameters.

A Field-Scale Approach to Estimate Nitrate Loading to Groundwater.

The quantification of groundwater NO loading associated with a specific field or set of management practices so that groundwater quality improvements can be objectively assessed is a major challenge. The magnitude and timing of NO export from a single agricultural field under raspberry ( L.) production were investigated by combining high-resolution groundwater NO concentration profiles (sampled using passive diffusion samplers) with Darcy's flux estimation at the field's down-gradient edge (based on field-measured hydraulic gradients and laboratory-estimated hydraulic conductivity). Annual recharge estimated using Darcy's law (1002 mm) was similar to that obtained using two other approaches. The similarity in the rate of Cl applied to the field and the estimated export flux over the 1-yr monitoring period (51 vs. 56 kg Cl ha) suggested the mass flux estimation approach was robust. An estimated 80 kg NO-N ha was exported from the agricultural field over the 1-yr monitoring period. The greatest monthly groundwater mass flux exported was observed in February and March (∼11 kg NO-N ha), and was associated with NO leached from the soil zone during the onset of precipitation in the previous autumn. Provided the groundwater recharged from the field of interest can be isolated within a vertical profile, this approach is an effective method for obtaining spatially integrated estimates of the magnitude and timing of NO loading to groundwater.

Pathogenic Streptomyces spp. Abundance Affected by Potato Cultivars.

Potato cultivars vary in their tolerance to common scab; however, how they affect common scab-causing Streptomyces spp. populations over time is poorly understood. This study investigated the effects of potato cultivar on pathogenic Streptomyces spp. abundance, measured using quantitative PCR, in three spatial locations in a common scab-infested field: (i) soil close to the plant (SCP); (ii) rhizosphere soil (RS); and (iii) geocaulosphere soil (GS). Two tolerant (Gold Rush, Hindenburg) and two susceptible cultivars (Green Mountain, Agria) were tested. The abundance of pathogenic Streptomyces spp. significantly increased in late August compared with other dates in RS of susceptible cultivars in both years. Abundance of pathogenic Streptomyces spp., when averaged over locations and time, was significantly greater in susceptible cultivars compared with tolerant cultivars in 2014. Principal coordinates analysis showed that SCP and RS soil properties (pH, organic carbon, and nitrogen concentrations) explained 68 and 76% of total variation in Streptomyces spp. abundance among cultivars in 2013, respectively, suggesting that cultivars influenced common scab pathogen growth conditions. The results suggested that the genetic background of potato cultivars influenced the abundance of pathogenic Streptomyces spp., with five to six times more abundant Streptomyces spp. in RS of susceptible cultivars compared with tolerant cultivars, which would result in substantially more inoculum left in the field after harvest.

Use of δ15N and δ18O Values for Nitrate Source Identification under Irrigated Crops: A Cautionary Vadose Zone Tale.

Source nitrogen (N) identification of leachate or groundwater nitrate is complicated by N source mixing and N and oxygen (O) isotope fractionation caused by microbial N transformations. This experiment examined the δN and δO values in leachate collected over 1 yr at 55 cm below raspberry ( L.) plots receiving either synthetic fertilizer (FT) or poultry manure (MT). The large ranges of δN (FT: -2.4 to +8.7‰, MT: +1.6 to +9.6‰) and δO (FT: -9.9 to -0.3‰, MT: -10.9 to +1.7‰) values in leachate collected under crop rows prohibited the reliable identification of the applied N sources on individual sampling dates. However, the mass-weighted average δN (FT: +3.2‰, MT: +7.3‰) values in leachate were significantly different and can be explained by accounting for the estimated contributions of nitrate and δN values of the various N sources, including applied fertilizer (-0.7‰) or manure (+7.9‰), nitrate-rich irrigation water (+9.0‰), and nitrate from soil N mineralization and nitrification (FT: +3.7‰, MT: +4.6‰; the seasonal timing of which is unknown). This study illustrates the importance of characterizing all major N sources and considering the seasonal variation of these sources and of N cycling processes, as they contribute to the δN values of leachate.

Litter decay controlled by temperature, not soil properties, affecting future soil carbon.

Widespread global changes, including rising atmospheric CO2 concentrations, climate warming and loss of biodiversity, are predicted for this century; all of these will affect terrestrial ecosystem processes like plant litter decomposition. Conversely, increased plant litter decomposition can have potential carbon-cycle feedbacks on atmospheric CO2 levels, climate warming and biodiversity. But predicting litter decomposition is difficult because of many interacting factors related to the chemical, physical and biological properties of soil, as well as to climate and agricultural management practices. We applied 13 C-labelled plant litter to soil at ten sites spanning a 3500-km transect across the agricultural regions of Canada and measured its decomposition over five years. Despite large differences in soil type and climatic conditions, we found that the kinetics of litter decomposition were similar once the effect of temperature had been removed, indicating no measurable effect of soil properties. A two-pool exponential decay model expressing undecomposed carbon simply as a function of thermal time accurately described kinetics of decomposition. (R2  = 0.94; RMSE = 0.0508). Soil properties such as texture, cation exchange capacity, pH and moisture, although very different among sites, had minimal discernible influence on decomposition kinetics. Using this kinetic model under different climate change scenarios, we projected that the time required to decompose 50% of the litter (i.e. the labile fractions) would be reduced by 1-4 months, whereas time required to decompose 90% of the litter (including recalcitrant fractions) would be reduced by 1 year in cooler sites to as much as 2 years in warmer sites. These findings confirm quantitatively the sensitivity of litter decomposition to temperature increases and demonstrate how climate change may constrain future soil carbon storage, an effect apparently not influenced by soil properties.

The nitrogen responsive transcriptome in potato (Solanum tuberosum L.) reveals significant gene regulatory motifs.

Nitrogen (N) is the most important nutrient for the growth of potato (Solanum tuberosum L.). Foliar gene expression in potato plants with and without N supplementation at 180 kg N ha(-1) was compared at mid-season. Genes with consistent differences in foliar expression due to N supplementation over three cultivars and two developmental time points were examined. In total, thirty genes were found to be over-expressed and nine genes were found to be under-expressed with supplemented N. Functional relationships between over-expressed genes were found. The main metabolic pathway represented among differentially expressed genes was amino acid metabolism. The 1000 bp upstream flanking regions of the differentially expressed genes were analysed and nine overrepresented motifs were found using three motif discovery algorithms (Seeder, Weeder and MEME). These results point to coordinated gene regulation at the transcriptional level controlling steady state potato responses to N sufficiency.

Novel P450nor Gene Detection Assay Used To Characterize the Prevalence and Diversity of Soil Fungal Denitrifiers.

UNLABELLED: Denitrifying fungi produce nitrous oxide (N2O), a potent greenhouse gas, as they generally lack the ability to convert N2O to dinitrogen. Contrary to the case for bacterial denitrifiers, the prevalence and diversity of denitrifying fungi found in the environment are not well characterized. In this study, denitrifying fungi were isolated from various soil ecosystems, and novel PCR primers targeting the P450nor gene, encoding the enzyme responsible for the conversion of nitric oxide to N2O, were developed, validated, and used to study the diversity of cultivable fungal denitrifiers. This PCR assay was also used to detect P450nor genes directly from environmental soil samples. Fungal denitrification capabilities were further validated using an N2O gas detection assay and a PCR assay targeting the nirK gene. A collection of 492 facultative anaerobic fungi was isolated from 15 soil ecosystems and taxonomically identified by sequencing the internal transcribed spacer sequence. Twenty-seven fungal denitrifiers belonging to 10 genera had the P450nor and the nirK genes and produced N2O from nitrite. N2O production is reported in strains not commonly known as denitrifiers, such as Byssochlamys nivea, Volutella ciliata, Chloridium spp., and Trichocladium spp. The prevalence of fungal denitrifiers did not follow a soil ecosystem distribution; however, a higher diversity was observed in compost and agricultural soils. The phylogenetic trees constructed using partial P450nor and nirK gene sequences revealed that both genes clustered taxonomically closely related strains together. IMPORTANCE: A PCR assay targeting the P450nor gene involved in fungal denitrification was developed and validated. The newly developed P450nor primers were used on fungal DNA extracted from a collection of fungi isolated from various soil environments and on DNA directly extracted from soil. The results indicated that approximatively 25% of all isolated fungi possessed this gene and were able to convert nitrite to N2O. All soil samples from which denitrifying fungi were isolated also tested positive for the presence of P450nor The P450nor gene detection assay was reliable in detecting a large diversity of fungal denitrifiers. Due to the lack of homology existing between P450nor and bacterial denitrification genes, it is expected that this assay will become a tool of choice for studying fungal denitrifiers.

Tillage Management and Seasonal Effects on Denitrifier Community Abundance, Gene Expression and Structure over Winter.

Tillage effects on denitrifier communities and nitrous oxide (N2O) emissions were mainly studied during the growing season. There is limited information for the non-growing season, especially in northern countries where winter has prolonged periods with sub-zero temperatures. The abundance and structure of the denitrifier community, denitrification gene expression and N2O emissions in fields under long-term tillage regimes [no-tillage (NT) vs conventional tillage (CT)] were assessed during two consecutive winters. NT exerted a positive effect on nirK and nosZ denitrifier abundance in both winters compared to CT. Moreover, the two contrasting managements had an opposite influence on nirK and nirS RNA/DNA ratios. Tillage management resulted in different denitrifier community structures during both winters. Seasonal changes were observed in the abundance and the structure of denitrifiers. Interestingly, the RNA/DNA ratios were greater in the coldest months for nirK, nirS and nosZ. N2O emissions were not influenced by management but changed over time with two orders of magnitude increase in the coldest month of both winters. In winter of 2009-2010, emissions were mainly as N2O, whereas in 2010-2011, when soil temperatures were milder due to persistent snow cover, most emissions were as dinitrogen. Results indicated that tillage management during the growing season induced differences in denitrifier community structure that persisted during winter. However, management did not affect the active cold-adapted community structure.

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.

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 nitrate and glucose addition on denitrification and nitric oxide reductase (cnorB) gene abundance and mRNA levels in Pseudomonas mandelii inoculated into anoxic soil.

The effect of glucose addition (0 and 500 µg C g(-1) soil) and nitrate (NO(3)) addition (0, 10, 50 and 500 µg NO(3)-N g(-1) soil) on nitric oxide reductase (cnorB) gene abundance and mRNA levels, and cumulative denitrification were quantified over 48 h in anoxic soils inoculated with Pseudomonas mandelii. Addition of glucose-C significantly increased cnorB(p) (P. mandelii and related species) mRNA levels and abundance compared with soil with no glucose added, averaged over time and NO(3) addition treatments. Without glucose addition, cnorB(p) mRNA levels were higher when 500 µg NO(3)-N g(-1) soil was added compared with other NO(3) additions. In treatments with glucose added, addition of 50 µg NO(3)-N g(-1) soil resulted in higher cnorB(p) mRNA levels than soil without NO(3) but was not different from the 10 and 500 µg NO(3)-N g(-1) treatments. cnorB(p) abundance in soils without glucose addition was significantly higher in soils with 500 µg NO(3)-N g(-1) soil compared to lower N-treated soils. Conversely, addition of 500 µg NO(3)-N g(-1) soil resulted in lower cnorB(p) abundance compared with soil without N-addition. Over 48 h, cumulative denitrification in soils with 500 µg glucose-C g(-1) soil, and 50 or 500 µg NO(3)-N g(-1) was higher than all other treatments. There was a positive correlation between cnorB(p) abundance and cumulative denitrification, but only in soils without glucose addition. Glucose-treated soils generally had higher cnorB(p) abundance and mRNA levels than soils without glucose added, however response of cnorB(p) abundance and mRNA levels to NO(3) supply depended on carbon availability.

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

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 (N(2)O) 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 (nirS(P)) increased only in glucose-amended soil: the nirS(P) 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 x 10(7) gene copies and 6.9 x 10(7) transcripts g(-1) dry soil. Cumulative N(2)O emissions and denitrification rates increased over 72 h in both glucose- and plant-tissue-C-treated soil. The nirS(P) 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.

Effect of nitrate and acetylene on nirS, cnorB, and nosZ expression and denitrification activity in Pseudomonas mandelii.

Nitrate acts as an electron acceptor in the denitrification process. The effect of nitrate in the range of 0 to 1,000 mg/liter on Pseudomonas mandelii nirS, cnorB, and nosZ gene expression was studied, using quantitative reverse transcription-quantitative PCR. Denitrification activity was measured by using the acetylene blockage method and gas chromatography. The effect of acetylene on gene expression was assessed by comparing denitrification gene expression in P. mandelii culture grown in the presence or absence of acetylene. The higher the amount of NO(3)(-) present, the greater the induction and the longer the denitrification genes remained expressed. nirS gene expression reached a maximum at 2, 4, 4, and 6 h in cultures grown in the presence of 0, 10, 100, and 1,000 mg of KNO(3)/liter, respectively, while induction of nirS gene ranged from 12- to 225-fold compared to time zero. cnorB gene expression also followed a similar trend. nosZ gene expression did not respond to NO(3)(-) treatment under the conditions tested. Acetylene decreased nosZ gene expression but did not affect nirS or cnorB gene expression. These results showed that nirS and cnorB responded to nitrate concentrations; however, significant denitrification activity was only observed in culture with 1,000 mg of KNO(3)/liter, indicating that there was no relationship between gene expression and denitrification activity under the conditions tested.

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

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 (N(2)O) 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 micromol, 63 micromol, and 22 micromol at pH 6, 7, and 8, respectively, at 8 h, whereas negligible denitrification was measured at pH 5. P. mandelii cells grown at 20 degrees C and 30 degrees 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 degrees C did not occur in the first 4 h. Levels of cumulative denitrification at 10 h were 6.6 micromol for P. mandelii cells grown at 10 degrees C and 20 degrees C and 30 micromol for cells grown at 30 degrees 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 degrees C. Low pH and temperature negatively affected denitrification activity.

Nitric oxide reductase gene expression and nitrous oxide production in nitrate-grown Pseudomonas mandelii.

Pure cultures of Pseudomonas mandelii were incubated with or without nitrate, which acts as a substrate and an electron acceptor for denitrification. Nitric oxide reductase (cnorB) gene expression was measured using a quantitative reverse transcription-PCR, and nitrous oxide emissions were measured by gas chromatography. P. mandelii cells in either the presence or absence of nitrate demonstrated an increase in cnorB gene expression during the first 3 h of growth. The level of expression of cnorB in nitrate-amended cells remained high (average, 2.06 x 10(8) transcripts/microg of RNA), while in untreated cells it decreased to an average of 3.63 x 10(6) transcripts/microg of RNA from 4 to 6 h. Nitrous oxide accumulation in the headspace was detected at 2 h, and cumulative emissions continued to increase over a 24-h period to 101 mumol in nitrate-amended cells. P. mandelii cnorB gene expression was not detected under aerobic conditions. These results demonstrate that P. mandelii cnorB gene expression was induced 203-fold at 4 h when nitrate was present in the medium. Accumulations of N(2)O indicated that the cNorB enzyme was synthesized and active.

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

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 (H(p)), and in the "furrow" (F). The denitrification rates, N(2)O 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 cnorB(B) guilds), whereas the cnorB(P) community (Pseudomonas mandelii and closely related spp.) showed an approximately 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 cnorB(P) 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 cnorB(B) gene numbers. Gene numbers were higher for the H(p) than the H location for the nosZ and nirK communities and for the cnorB(P) community on individual dates, presumably indicating an effect of the plant on denitrifier abundance. Higher cnorB(P) gene numbers for the H location than the F location and for nosZ and cnorB(B) 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 cnorB(P) gene numbers, extractable organic carbon values, and temperature. Denitrification and N(2)O emissions were mostly regulated by inorganic nitrogen availability and water-filled pore space but were uncoupled from denitrifier community abundances measured in this system.