Azospirillum inoculation and nitrogen fertilization effect on grain yield and on the diversity of endophytic bacteria in the phyllosphere of rice rainfed crop

We assessed the Azospirillum inoculation and N-fertilization effect on grain yield and on the phyllosphere endophytic diversity of nitrogen-fixing bacteria in a rice rainfed crop. We used cultivation-based techniques and cultivation-independent methods involving PCR-16S rRNA and denaturing gradient gel electrophoresis (DGGE). In general, we observed that grain yield was improved when inoculated with Azospirillum (depending on the genotype) and/or fertilized with urea. A similar behavior was observed in total N-content in grain and the MPN determination, as the highest values occurred when seeds were inoculated with A. brasilense REC3 (S1) than with A brasilense 13-2C (S2). A positive nitrogenase activity and PCR-nifH amplification suggests that the bacteria associated to inner tissues of rice phyllosphere could have contributed to the different N-contents detected. The bacterial diversity, observed in the number and intensity of DGGE profiles, showed a higher number of bands when total DNA was obtained using only CTAB than with CTAB + PVP. The DGGE profiles revealed great stability in the dominating bands, which presumably represent numerically dominant species. Application of A. brasilense strains as inoculants did not influence the dominant members of the endophytic microbial communities in the phyllosphere, but improved N-content and production of rainfed rice crop.     

Implications of Azospirillum brasilense inoculation and nutrient addition on maize in soils of the Brazilian Cerrado under greenhouse and field conditions

The combination of nitrogen and Azospirillum can ensure greater nutrient absorption and crop yield in agricultural areas using high technology. Thus, the objective was to evaluate maize response to Azopirillum brasilense (AZ) inoculation and nutrient (macronutrients and micronutrients) application under greenhouse and field conditions in clay and sandy soils of the Brazilian Cerrado. In the greenhouse assays, the following parameters were measured: shoot dry weight (SDW), root dry weight (RDW), and root volume (RV). In the field experiments, the maize yield was determined after drying the grains at 60 °C for 48 h. In clay soil, there was a significant increase in the SDW, RDW and RV in the treatment with AZ concentrated (10¹¹ cells ml⁻¹ of inoculum) when compared with the control treatment and the treatment with AZ diluted (10⁶ cells ml⁻¹ of inoculum). In this soil, adding micronutrients did not affect the maize response under greenhouse conditions. In sandy soil, there was no difference between the AZ treatment and the control, except for treatments where nutrients and AZ were both added leading to a significant increase in the maize response. In both soils, the RV:RDW ratio was higher in the treatment with AZ concentrated compared to that in the treatment with AZ diluted, but the yield response depended on the addition of nutrients. Inoculation with A. brasilense gave comparable yield to the nitrogen treatment. The grain production was increased by 29% in the treatment with A. brasilense and nitrogen compared to nitrogen fertilization alone. In this study, the yield response was affected significantly when maize was inoculated with A. brasilense, but this response was dependent on the soil type under greenhouse conditions.     

Cadaverine production by Azospirillum brasilense and its possible role in plant growth promotion and osmotic stress mitigation

Polyamines are considered as plant growth regulating compounds; among them, cadaverine has been correlated with root growth promotion or osmotic stress mitigation in some plant species. The purpose of this study was to evaluate the capacity of bacterial Azospirillum brasilense Az39 strain to produce cadaverine in chemically defined medium and inoculated plants, and to correlate this capacity with root growth promotion or osmotic stress mitigation in hydroponics conditions. To evaluate cadaverine production in chemically defined medium A. brasilense Az39 was cultivated aerobically at 30 °C and 80 rpm in NFb medium or NFb-l supplemented with the precursor l-lysine. To evaluate the bacterial cadaverine production and growth promotion in plants, rice (Oryza sativa L.) cv. El Paso 144 seedlings were inoculated and hydroponically cultured under optimal conditions in growth chamber. In both, cadaverine was identified and quantified by dansyl-derivative method using a fluorescence-HPLC system, and lysine decarboxylase (LDC) activity was determined by ¹⁴CO₂ production in a closed tube system fed with [¹⁴C]-lysine. To evaluate the possible role of bacterial cadaverine in osmotic stress conditions, abscisic acid (ABA) production was analyzed in rice seedlings hydroponically cultured under 0 (no stress), ?0.47 (stress) or ?0.82 (severe stress) MPa osmotic potential generated by mannitol, with the addition of 1 nM or 1 μM cadaverine or A. brasilense Az39 inoculation. Our results indicate that A. brasilense Az39 promoted root growth and helped mitigate osmotic stress in rice seedlings, due in part to cadaverine production.     

Seedlings growth promotion by Azospirillum brasilense under normal and drought conditions remains unaltered in Tebuconazole-treated wheat seeds

The wide use of pesticides in modern agriculture may cause side effects on the non-target microflora. Data on the fungicide Tebuconazole effects on Azospirillum-wheat association are scarce. We analyzed the effects of Tebuconazole on: (a) Azospirillum brasilense Sp245 growth in pure culture, (b) A. brasilense Sp245 colonization of Triticum aestivum cv ProINTA Oasis roots, (c) A. brasilense Sp245-inoculated seedlings growth under normal and water stress conditions in the presence of 20% polyethylene glycol 8000. Seeds were separated in Tebuconazole-free and Tebuconazole-treated lots. Inoculated and non-inoculated seedlings were grown in hydroponics in the dark at 20 °C for 72 h. Root surface, coleoptile length, fresh and dry (DW) weights in both tissues and diazotrophic bacterial most probable number in roots were determined. Water contents and shoot-to-roots DW ratio were calculated. Neither Azospirillum growth nor root colonization was affected by Tebuconazole. Under normal growth conditions most of the growth parameters analyzed, revealed a clear positive effect of A. brasilense on wheat seedlings up to 72 h treatments. The characteristic Azospirillum enhancing effects observed on roots remained unaltered by Tebuconazole. The present study shows that Tebuconazole is compatible with A. brasilense Sp245-wheat inoculation.     

Factors affecting transfer of degradative plasmids between bacteria in soils

Horizontal gene transfer is useful for enhancing bioremediation through gene bioaugmentation. However, factors affecting transfer of degradative plasmids have not been systematically addressed. To this end, plasmid transfer experiments were performed using a TOL-like plasmid carrying the gene encoding for catechol 2,3-dioxygenase (C23O) between two soil bacteria under different conditions. Transfer frequency increased with air temperature in the range of 10–35 °C and reached 6 × 10⁻⁴ transconjugants per donor cell at 35 °C. The transfer frequency detected at soil depth 5–10 cm was significantly higher (p < 0.05) compared with other depths. Addition of 5–75% LB in the microbial inoculum promoted plasmid transfer frequencies. Addition of phenol to the experimental system resulted in significantly higher transfer frequency (p < 0.05) compared with no addition. Transfer frequency heat-moisture in loam was significantly higher (p < 0.05) than in other soils. The highest transfer frequency was found in the experiment containing tomato seedlings, with up to about 1.3 × 10⁻³ transconjugants per donor cell. Corn and wheat seedlings also led to significantly higher transfer frequencies (p < 0.05) compared with no plants. Furthermore, C23O activities of transconjugants formed under different conditions were measured, as a surrogate measure of the activity of transconjugant. Transfer temperature, soil and plant types had a minor influence on activities of transconjugants. Topsoil (0–5 cm) transconjugants expressed C23O more efficiently under normal incubation condition, but less efficiently when soils incubated with excessive LB medium concentrations, and in the absence of phenol in soil. These results suggested that transfer temperature, soil depth, dilutions of LB broth, phenol content, and soil and plant types had important effects on transfer of the TOL-like plasmid in soil, and some factors also affected activities of transconjugants.     

Field performance of a liquid formulation of Azospirillum brasilense on dryland wheat productivity

The beneficial effects of inoculating with Azospirillum brasilense on crop productivity have been widely described, but extensive use in typical agricultural field environments is scarcely documented. The objective of this study was to quantify the productivity of wheat (Triticum aestivum L.) whose seed was inoculated with a liquid formulation containing Azospirillum brasilense INTA Az-39 strain under typical dryland farming conditions. The study was performed in the 2002–2006 growing seasons, evaluating inoculated and non-inoculated seed at 297 experimental locations in the Pampas region of Argentina. The inoculated crops exhibited more vigorous vegetative growth, with both greater shoot and root dry matter accumulation (12.9 and 22.0%, respectively). The inoculation increased the number of harvested grains by 6.1%, and grain yield by 260 kg ha^?1 (8.0%). Positive responses were determined in about 70% of the sites, depending mostly on the attainable yield and independently of fertilization and other crop and soil management practices. In general, more response to inoculation was observed in the absence of major crop growth limitations, suggesting the complementary contribution of the Azospirillum brasilense treatment to more efficiently developing higher yielding wheat.     

Fate of sulfadiazine administered to pigs and its quantitative effect on the dynamics of bacterial resistance genes in manure and manured soil

The veterinary antibiotic sulfadiazine (SDZ), labelled by ¹⁴C, was administered to pigs to follow the fate of the drug and its metabolites in manure and manure-amended soil, and to investigate the dynamics of drug effects on resistance genes and bacterial communities. In the manure sampled over 10 days, more than 96% of the drug was found as parent compound or metabolites N-acetyl-SDZ and 4-hydroxy-SDZ. While the manure was stored the concentration of SDZ increased by 42% due to deacetylation of the metabolite N-acetyl-SDZ, whereas the minor metabolite 4-hydroxy-SDZ kept constant. In the soil the extractable amounts of the compounds decreased exponentially to less than 1 mg kg^?1 within 11 days after manure amendment. The abundances of SDZ resistance genes sul1 and sul2 were determined by qPCR relative to 16S rRNA genes in total DNA from manure and manure-amended soil. In manure both genes increased exponentially in copy number during the first 60 days of storage, suggesting preferential growth of resistant populations. However, the abundance of sul1 and sul2 decreased below 10^?5 copies per 16S rRNA gene after 175 days. With manure high amounts of sul1 and sul2 were introduced into the soil which were reduced by more than 10 times within 24 days. Thereafter, sul1 was stably maintained in soil, while sul2 further decreased between day 60 and day 165. A mathematical model was developed that could well explain the time course of sul gene abundance by considering the cost of sul genes, horizontal gene transfer, and selection of the resistant populations in the presence of SDZ. Modelling revealed a selective effect of SDZ on sul2 even at low concentrations down to 0.15 mg kg^?1 soil. Bacterial community profiles of manure and manure-amended soil were distinct, indicating that bacteria introduced with manure do not become prominent in soil. The composition of the bacterial community in manure constantly changed during storage, but mainly during the first 10 days. Profiles of soil bacterial communities revealed only a transient perturbation by manure containing SDZ.     

Influence of carbon amendments on soil denitrifier abundance in soil microcosms

It is known that carbon (C) amendments increase microbial activity in anoxic soil microcosm studies, however the effects on abundance of total and denitrifier bacterial communities is uncertain. Quantitative PCR was used to target the 16S rRNA gene for the total bacterial community, the nosZ functional gene to reflect a broad denitrifier community, and functional genes from narrow denitrifier communities represented by Pseudomonas mandelii and related species (cnorB_P) and Bosea/Bradyrhizobium/Ensifer spp. (cnorB_B). Repacked soil cores were amended with varying amounts of glucose and red clover plant tissue (0–1000 mg C kg^? 1 of soil) and incubated for 96 h. Carbon amendment significantly increased respiration as measured by cumulative CO₂ emissions. Inputs of red clover or glucose at 1000 mg C kg^? 1 of soil caused increased abundance in the total bacteria under the conditions used. There was about an approximate 2-fold increase in the abundance of bacteria bearing the nosZ gene, but only in treatments receiving 500 or 1000 mg C kg^? 1 of soil of glucose or red clover, respectively. Additions of ≥ 500 mg C kg^? 1 soil of red clover and ≥ 250 mg C kg^? 1 of glucose increased cnorB_P-gene bearing denitrifiers. Changes in abundance of the targeted communities were related to C availability in soil, as indicated by soil respiration, regardless of C source. Applications of C amendments at rates that would occur in agricultural soils not only increase microbial activity, but can also induce changes in abundance of total bacterial and denitrifier communities in studies of anoxic soil microcosms.     

Bacterial community structure of nirK-bearing denitrifiers and the development of properties of soils in created mitigation wetlands

We investigated the abundance and genetic heterogeneity of bacterial nitrite reductase genes (nir) and soil structural properties in created and natural freshwater wetlands in the Virginia piedmont. Soil attributes included soil organic matter (SOM), total organic carbon (TOC), total nitrogen (TN), pH, gravimetric soil moisture (GSM), and bulk density (D_b). A subset of soil attributes were analyzed across the sites, using euclidean cluster analysis, resulting in three soil condition (SC) groups of increasing wetland soil development (i.e., SC1 < SC2 < SC3; less to more developed or matured) as measured by accumulation of TOC, TN, the increase of GSM, and the decrease of D_b. There were no difference found in the bacterial community diversity between the groups (p = 0.4). NirK gene copies detected ranged between 3.6 × 10⁴ and 3.4 × 10⁷ copies g⁻¹ soil and were significantly higher in the most developed soil group, SC3, than in the least developed soil group, SC1. However, the gene copies were lowest in SC2 that had a significantly higher soil pH (~6.6) than the other two SC groups (~5.3). The same pattern was found in denitrifying enzyme activity (DEA) on a companion study where DEA was found negatively correlated with soil pH. Gene fragments were amplified and products were screened by terminal restriction fragment length polymorphism (T-RFLP) analysis. Among 146 different T-RFs identified, fourteen were dominant and together made up more than 65% of all detected fragments. While SC groups did not relate to whole nirK communities, most soil properties that identified SC groups did significantly correlate to dominant members of the community.     

Climatic influences on active fractions of soil organic matter

Biologically active fractions of soil organic matter are important in understanding decomposition potential of organic materials, nutrient cycling dynamics, and biophysical manipulation of soil structure. We evaluated the quantitative relationships among potential C and net N mineralization, soil microbial biomass C (SMBC), and soil organic C (SOC) under four contrasting climatic conditions. Mean SOC values were 28±11 mg g⁻¹ (n=24) in a frigid–dry region (Alberta/British Columbia), 25±5 mg g⁻¹ (n=12) in a frigid–wet region (Maine), 11±4 mg g⁻¹ (n=117) in a thermic–dry region (Texas), and 12±5 mg g⁻¹ (n=131) in a thermic–wet region (Georgia). Higher mean annual temperature resulted in consistently greater basal soil respiration (1.7 vs 0.8 mg CO₂–C g⁻¹ SOC d⁻¹ in the thermic compared with the frigid regions, P<0.001), greater net N mineralization (2.8 vs 1.3 mg inorganic N g⁻¹ SOC 24 d⁻¹, P<0.001), and greater SMBC (53 vs 21 mg SMBC g⁻¹ SOC, P<0.001). Specific respiratory activity of SMBC was, however, consistently lower in the thermic than in the frigid regions (29 vs 34 mg CO₂–C g⁻¹ SMBC d⁻¹, P<0.01). Higher mean annual precipitation resulted in consistently lower basal soil respiration (1.1 vs 1.3 mg CO₂–C g⁻¹ SOC d⁻¹ in the wet compared with the dry regions, P<0.01) and lower SMBC (31 vs 43 mg SMBC g⁻¹ SOC, P<0.001), but had inconsistent effects on net N mineralization that depended upon temperature regime. Specific respiratory activity of SMBC was consistently greater in the wet than the dry regions (≈33 vs 29 mg CO₂–C g⁻¹ SMBC d⁻¹, P<0.01). Although the thermic regions were not able to retain as high a level of SOC as the frigid regions, due likely to high annual decomposition rates, biologically active soil fractions were as high per mass of soil and even 2–3-times greater per unit of SOC in the thermic compared with the frigid regions. These results suggest that macroclimate has a large impact on the portion of soil organic matter that is potentially active, but a relatively small impact on the specific respiratory activity of SMBC.     

Detection and quantification of the nematophagous fungus Hirsutella minnesotensis in soil with real-time PCR

A real-time PCR assay was developed to quantify in soil the fungus Hirsutella minnesotensis, an important parasite of secondary-stage juvenile (J2) of the soybean cyst nematode. A primer pair 5′-GGGAGGCCCGGTGGA-3′ and 5′-TGATCCGAGGTCAACTTCTGAA-3′ and a TaqMan probe 5′-CGTCCGCCGTAAAACGCCCAAC-3′ were designed based on the sequence of the ITS region of the rRNA gene. The primers were highly species-specific. The PCR reaction system was very sensitive and able to detect as few as 4 conidia g^?1 soil. Regression analysis showed similar slopes and efficiency on DNA from pure culture (y = ?3.587x + 41.017, R² = 0.9971, E = 0.9055) and from Log conidia g^?1 soil (y = ?3.855x + 37.669, R² = 0.9139, E = 0.8172), indicating that the real-time PCR protocol can reliably quantify H. minnesotensis in the soil. The real-time PCR assay was applied to 20 soil samples from soybean fields, and compared with a parasitism assay. The real-time PCR assay detected H. minnesotensis in six of the soils, whereas the parasitism assay detected H. minnesotensis in the same six soils and three additional soils. The real-time PCR assay was weakly correlated (R² = 0.49) with the percentage of parasitized J2 in the six soils, indicating that different types of soil may interfere the efficiency of the real-time PCR assay, possibly due to the effect of soil types on efficacy of DNA extraction. The parasitism assay appeared to be more sensitive than real-time PCR in detecting presence of H. minnesotensis, but real-time PCR was much faster and less costly and provided a direct assessment of fungal biomass. Using the two assays in combination can obtain more complete information about the fungus in soil than either assay alone. Hirsutella parasitism was widespread and detected in 13 of the 20 field soils, indicating that these fungi may contribute to suppressiveness of soybean cyst nematode in nature and likely have high biological control potential for the nematode.     

Isotopic evidence of significant assimilation of atmospheric-derived nitrogen fixed by Azospirillum brasilense co-inoculated with phosphate-solubilising Pantoea dispersa in pepper seedling

The contribution of plant growth-promoting bacteria (PGPB) of the genus Azospirillum to the plant N budget through biological nitrogen fixation (BNF) is still controversial. The aim of this study was to determine the contribution of BNF by Azospirillum brasilense on pepper grown at different N levels, attained using the ¹⁵N natural abundance method. To this end, pepper plants were grown in a growth chamber and treated with A. brasilense combined with Pantoea dispersa and then irrigated at four different N levels (0, 1, 3 and 7 mM NO₃⁻). The assimilation of fixed N was clear from the lower δ¹⁵N values observed in bacteria-treated plants compared with those of non-bacteria treated plants. The percentage of BNF-derived N decreased with decreasing NO₃⁻ levels in the growth medium. BNF contribution to the total nitrogen content of plants was found to be as high as 46%. The results suggest that the bacteria have a potential to supply a considerable amount of N to pepper seedlings, as well as to stimulate plant growth and N uptake when Azospirillum–Pantoea treatment is combined with low NO₃⁻ levels.     

Incongruous variation of denitrifying bacterial communities as soil N level rises in Canadian canola fields

Soil N fertilization stimulates the activity of the soil bacterial species specialized in performing the different steps of the denitrification processes. Different responses of these bacterial denitrifiers to soil N management could alter the efficiency of reduction of the greenhouse gas N₂O into N₂ gas in cultivated fields. We used next generation sequencing to show how raising the soil N fertility of Canadian canola fields differentially modifies the diversity and composition of nitrite reductase (nirK and nirS) and nitrous oxide reductase (nosZ) gene-carrying denitrifying bacterial communities, based on a randomized complete blocks field experiment. Raising soil N levels increased up to 60% the ratio of the nirK to nirS genes, the two nitrite reductase coding genes, in the Brown soil and up to 300% in the Black soil, but this ratio was unaffected in the Dark Brown soil. Raising soil N levels also increased the diversity of the bacteria carrying the nitrite reductase gene nirK (Simpson index, P = 0.0417 and Shannon index, 0.0181), and changed the proportions of the six dominant phyla hosting nirK, nirS, and nosZ gene-carrying bacteria. The level of soil copper (Cu) and the abundance of nirK gene, which codes for a Cu-dependent nitrite reductase, were positively related in the Brown (P = 0.0060, R² = 0.48) and Dark Brown (0.0199, R² = 0.59) soils, but not in the Black soil. The level of total diversity of the denitrifying communities tended to remain constant as N fertilization induced shifts in the composition of these denitrifying communities. Together, our results indicate that higher N fertilizer rate increases the potential risk of nitrous oxide (N₂O) emission from canola fields by promoting the proliferation of the mostly adaptive N₂O-producing over the less adaptive N₂O-reducing bacterial community.     

Lumbricus terrestris L. does not impact on the remediation efficiency of compost and biochar amendments

The aim of this study was to test the impact of compost and biochar, with or without earthworms, on the mobility and availability of metals, and on the growth of grass to re-vegetate contaminated soil from the Parys Mountain mining site, Anglesey. We also determined if the addition of earthworms compromises remediation efforts.In a laboratory experiment, contaminated soil (1343 mg Cu kg^?1, 2511 mg Pb kg^?1 and 262 mg Zn kg^?1) was remediated with compost and/or biochar. After 77 days Lumbricus terrestris L. earthworms were added to the treatment remediated with both compost and biochar, and left for 28 days. L. terrestris was not able to survive in the biochar, compost or unamended treatments. A germination and growth bioassay, using Agrostis capillaris (Common Bent) was then run on all treatments for 28 days.The combination of biochar and compost decreased water soluble Cu (from 5.6 to 0.2 mg kg^?1), Pb (from 0.17 to less than 0.007 mg kg^?1) and Zn (from 3.3 to 0.05 mg kg^?1) in the contaminated soil and increased the pH from 2.7 to 6.6. The addition of L. terrestris to this treatment had no effect on the concentration of the water soluble metals in the remediated soil.The compost was the only treatment that resulted in germination and growth of A. capillaris suitable for re-vegetation purposes. However, the combination of compost and biochar (with or without L. terrestris) produced the lowest concentrations of Cu (8 mg kg^?1) and Zn (36 mg kg^?1) in the aboveground biomass, lower than the compost treatment (15 mg Cu kg^?1 and 126 mg Zn kg^?1).The addition of biochar and compost both separately and as co-amendments was effective in reducing the mobility and availability of metals. The addition of L. terrestris did not re-mobilise previously sequestered metals.     

Isotope ratio mass spectrometry identifies soil microbial biocontrol agents having trophic relations with the plant pathogen Armillaria mellea

An understanding of the types of interactions that take place between plant pathogens and other microorganisms in the natural environment is crucial in order to identify new potential biocontrol agents. The use of microorganisms labelled with stable isotopes is a potentially useful method for studying direct parasitisation of a given pathogen or assimilation of the pathogen's metabolites by microorganisms. A microorganism labelled with a stable isotope can be monitored in the environment and isotope ratio mass spectrometry can detect whether it is directly parasitised or its metabolites are used by other microorganisms. In this study, we isolated 158 different species of fungi and bacteria from soil and assayed their biocontrol potential against a plant pathogen (Armillaria mellea) by coupling a dual-culture test with mass spectrometry analysis of the ¹³C isotope in the microorganisms in presence of ¹³C-labelled A. mellea. The microorganisms affected the pathogen by means of antibiosis phenomena (total or partial inhibition of pathogen growth, alteration of its morphology) and by antagonism, probably resulting from competition for space and nutrients or from mycoparasitism. Isotope ratio mass spectrometry was used to identify direct trophic interactions between microorganisms and the pathogen as in dual cultures as in soil microcosms. Six fungi and one bacterium were found to display the best active trophic behaviour against the pathogenin dual cultures; three microorganisms were discarded due to their plant pathogen potential. Trichoderma harzianum, Pseudomonas fluorescens and Rhodosporidium babjevae were selected to carry out the experiments. T. harzianum inhibited pathogen development (rate of inhibition 80 ± 0.19%) and its δ ¹³C values increased (244.03 ± 36.70‰) in contact with ¹³C-labelled A. mellea. Lower levels of antagonism and correspondingly lower assimilation of ¹³C were detected in P. fluorescens and R. babjevae. Only T. harzianum maintained mycoparasitic activity in the soil microcosm, showing a δ ¹³C value of 1.97 ± 2.24‰ after one month in co-presence with the labelled pathogen. This study provides support for the use of isotope ratio mass spectrometry as an additional tool in screening for potential biocontrol agents.     

Comparison of the eddy covariance and automated closed chamber methods for evaluating nocturnal CO2 exchange in a Japanese cypress forest

Underestimation of nocturnal CO₂ respiration using the eddy covariance method under calm conditions remains an unsolved problem at many flux observation sites in forests. To evaluate nocturnal CO₂ exchange in a Japanese cypress forest, we observed CO₂ flux above the canopy (F_c), changes in CO₂ storage in the canopy (S_t) and soil, and trunk and foliar respiration for 2 years (2003–2004). We scaled these chamber data to the soil, trunk, and foliar respiration per unit of ground area (F_s, F_t, F_f, respectively) and used the relationships of F_s, F_t, and F_f with air or soil temperature for comparison with canopy-scale CO₂ exchange measurements (=F_c + S_t). The annual average F_s, F_t, and F_f were 714 g C m⁻² year⁻¹, 170 g C m⁻² year⁻¹, and 575 g C m⁻² year⁻¹, respectively. At small friction velocity (u_*), nocturnal F_c + S_t was smaller than F_s + F_t + F_f estimated using the chamber method, whereas the two values were almost the same at large u_*. We replaced F_c + S_t measured during calm nocturnal periods with a value simulated using a temperature response function derived during well-mixed nocturnal periods. With this correction, the estimated net ecosystem exchange (NEE) from F_c + S_t data ranged from −713 g C m⁻² year⁻¹ to −412 g C m⁻² year⁻¹ in 2003 and from −883 g C m⁻² year⁻¹ to −603 g C m⁻² year⁻¹ in 2004, depending on the u_* threshold. When we replaced all nocturnal F_c + S_t data with F_s + F_t + F_f estimated using the chamber method, NEE was −506 g C m⁻² year⁻¹ and −682 g C m⁻² year⁻¹ for 2003 and 2004, respectively.     

Effects of inorganic fertilizer and manure on soil archaeal abundance at two experimental farms during three consecutive rotation-cropping seasons

Soil archaeal population dynamics at two experimental sites of the same clay-loam type in Ottawa and Woodslee, Ontario, were investigated to determine fertilizer and manure effects following their different long-term crop rotation and fertilization schemes. Phylogenetic analysis of cloned soil archaeal 16S rRNA gene libraries of both sites identified them with group 1.1b of Thaumarchaeota. The gene population dynamics subtly varied in the order of 10⁷ copies g⁻¹ soil when monitored by quantitative real-time PCR during three growing seasons (2007–2009). In Ottawa, where plots were amended with dairy-farm manure, soil thaumarchaeal gene abundance was double of the unamended plots. At the Woodslee N-P-K-fertilized plots, it remained at least 30% fewer than that of the unfertilized ones. These cultivated plots showed soil carbon limitation while the fertilized ones were low in soil pH (ca. 5.5). Surface soils from an unfertilized sod plot and an adjacent deciduous forest had higher total carbon content (C:N ratio of 9 and 11, respectively). Their thaumarchaeal gene abundance varied up to 4.8 × 10⁷ and 7.0 × 10⁷ copies g⁻¹ soil, respectively. The former value was also attained at the manure-amended plots in Ottawa, where the C:N ratio was just below 10. Where soil pH was above 6.0, there was a weak and positive correlation between soil total C and the estimated gene abundance. Such gene population dynamics consistently demonstrated the stimulating and suppressive effects of dairy-farm manure (Ottawa site) and inorganic fertilizers (Woodslee site), respectively, on soil thaumarchaea. At both sites archaeal amoA and 16S rRNA gene abundance were similarly affected. Archaeal amoA gene abundance also outnumbered bacterial amoA abundance, suggesting that ammonia-oxidizing archaea might be dominant in these soils. Only minor crop effects on gene population dynamics were detected.     

Evaluation of an optimal extraction method for measuring d-ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) in agricultural soils and its association with soil microbial CO2 assimilation

Assimilating atmospheric carbon (C) into terrestrial ecosystems is recognized as a primary measure to mitigate global warming. Ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) is the dominant enzyme by which terrestrial autotrophic bacteria and plants fix CO₂. To investigate the possibility of using RubisCO activity as an indicator of microbial CO₂ fixation potential, a valid and efficient method for extracting soil proteins is needed. We examined three methods commonly used for total soil protein extraction. A simple sonication method for extracting soil protein was more efficient than bead beating or freeze–thaw methods. Total soil protein, RubisCO activity, and microbial fixation of CO₂ in different agricultural soils were quantified in an incubation experiment using ¹⁴C-CO₂ as a tracer. The soil samples showed significant differences in protein content and RubisCO activity, defined as nmol CO₂ fixed g⁻¹ soil min⁻¹. RubisCO activities ranged from 10.68 to 68.07 nmol CO₂ kg⁻¹ soil min⁻¹, which were closely related to the abundance of cbbL genes (r = 0.900, P = 0.0140) and the rates of microbial CO₂ assimilation (r = 0.949, P = 0.0038). This suggests that RubisCO activity can be used as an indicator of soil microbial assimilation of atmospheric CO₂.     

Applying IUCN Red List criteria at a small regional level: A test case with butterflies in Flanders (north Belgium)

Red Lists are used to assess the extinction risk of species based on quantitative IUCN criteria. For the compilation of a new Red List of butterflies in Flanders (north Belgium), we collated ca 800,000 distribution records and applied the IUCN Red List criteria to this small region (ca 135,00 km²). We also explored the effect of spatial resolution on the outcome of the Red List assessment by alternatively using 1 × 1 km² and 5 × 5 km² grid cells for geographic range size and trend calculations. We determined conservation hot spots in Flanders based on the Red List status of the species composition in each grid cell. The new Red List classified 20 butterflies (out of 68 resident species) as Regionally Extinct, six as Critically Endangered, five as Endangered, seven as Vulnerable and seven as Near Threatened. The remaining 23 species were classified as Least Concern. Using coarse instead of fine grain grid cells would have classified ten species in a lower Red List category. Compared with the previous Red List, nine species were classified in a lower and 12 in a higher threat category. In total, 218 1 × 1 km² grid cells were considered as (very) high butterfly conservation priority sites. The application of the new IUCN criteria in a small region such as Flanders resulted in a Red List that offered challenging opportunities for the conservation of butterflies in particular and biodiversity in general.     

Comparative analysis of soil microbial communities and their responses to the short-term drought in bog,fen, and riparian wetlands

The frequency of drought is anticipated to increase in wetland ecosystems as global warming intensifies. However, information on microbial communities involved in greenhouse gas emissions and their responses to drought remains sparse. We compared the gene abundance of eubacterial 16S rRNA, nitrite reductase (nirS) and methyl coenzyme M reductase (mcrA), and the diversity and composition of eubacteria, methanogens and denitrifiers among bog, fen and riparian wetlands. The gene abundance, diversity and composition significantly differed among wetlands (p < 0.01) with the exception of the diversity of methanogens. The gene abundance was ranked in the order of the bog = fen > riparian wetland, whereas the diversity was in the riparian wetland ≥ fen > bog. In addition, we conducted a short-term drought experiment and compared microbial communities between control (water-logged) and drought (?15 cm) treatments. Drought led to significant decline in the gene abundance in the bog (16S rRNA, nirS, mcrA) (p < 0.01) and fen (16S rRNA, nirS) (p < 0.05), but not in the riparian wetland. There were no differences in the diversity and composition of denitrifiers and methanogens at all sites following drought. Our results imply that denitrifiers and methanogens inhabiting bogs and fens would suffer from short-term droughts, but remain unchanged in riparian wetlands.