The influence of alfalfa-switchgrass intercropping on microbial community structure and function.

The use of nitrogen fertilizer on bioenergy crops such as switchgrass results in increased costs, nitrogen leaching and emissions of N2 O, a potent greenhouse gas. Intercropping with nitrogen-fixing alfalfa has been proposed as an environmentally sustainable alternative, but the effects of synthetic fertilizer versus intercropping on soil microbial community functionality remain uncharacterized. We analysed 24 metagenomes from the upper soil layer of agricultural fields from Prosser, WA over two growing seasons and representing three agricultural practices: unfertilized switchgrass (control), fertilized switchgrass and switchgrass intercropped with alfalfa. The synthetic fertilization and intercropping did not result in major shifts of microbial community taxonomic and functional composition compared with the control plots, but a few significant changes were noted. Most notably, mycorrhizal fungi, ammonia-oxidizing archaea and bacteria increased in abundance with intercropping and fertilization. However, only betaproteobacterial ammonia-oxidizing bacteria abundance in fertilized plots significantly correlated to N2 O emission and companion qPCR data. Collectively, a short period of intercropping elicits minor but significant changes in the soil microbial community toward nitrogen preservation and that intercropping may be a viable alternative to synthetic fertilization.

Nitrous oxide emissions associated with ammonia-oxidizing bacteria abundance in fields of switchgrass with and without intercropped alfalfa.

The nitrogen (N) fertilizer required to supply a bioenergy industry with sufficient feedstocks is associated with adverse environmental impacts, including loss of oxidized reactive nitrogen through leaching and the production of the greenhouse gas nitrous oxide (N2 O). We examined effects on crop yield, N fate and the response of ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) to conventional fertilizer application or intercropping with N-fixing alfalfa, for N delivery to switchgrass (Panicum virgatum), a potential bioenergy crop. Replicated field plots in Prosser, WA, were sampled over two seasons for reactive nitrogen, N2 O gas emissions, and bacterial and archaeal ammonia monooxygenase gene (amoA) counts. Intercropping with alfalfa (70:30, switchgrass:alfalfa) resulted in reduced dry matter yields compared to fertilized plots, but three times lower N2 O fluxes (≤ 4 g N2 O-N ha-1 d-1 ) than fertilized plots (12.5 g N2 O-N ha-1 d-1 ). In the fertilized switchgrass plots, AOA abundance was greater than AOB abundance, but only AOB abundance was positively correlated with N2 O emissions, implicating AOB as the major producer of N2 O emissions. A life cycle analysis of N2 O emissions suggested the greenhouse gas emissions from cellulosic ethanol produced from switchgrass intercropped with alfalfa cultivation would be 94% lower than emissions from equivalent gasoline usage.

Affinity informs environmental cooperation between ammonia-oxidizing archaea (AOA) and anaerobic ammonia-oxidizing (Anammox) bacteria.

Anaerobic ammonia-oxidizing (Anammox) bacteria (AnAOB) rely on nitrite supplied by ammonia-oxidizing bacteria (AOB) and archaea (AOA). Affinities for ammonia and oxygen play a crucial role in AOA/AOB competition and their association with AnAOB. In this work we measured the affinity constants for ammonia and oxygen (half-saturation; km) of two freshwater AOA enrichments, an AOA soil isolate (N. viennensis), and a freshwater AnAOB enrichment. The AOA enrichments had similar kinetics (µmax ≈ 0.36 d-1, km,NH4 ≈ 0.78 µM, and km,O2 ≈ 2.9 µM), whereas N. viennensis had similar km values but lower µmax (0.23 d-1). In agreement with the current paradigm, these AOA strains showed a higher affinity for ammonia (lower km,NH4; 0.34-1.27 µM) than published AOB measurements (>20 µM). The slower growing AnAOB (µmax ≈ 0.16 d-1) had much higher km values (km,NH4 ≈ 132 µM, km,NO2 ≈ 48 µM) and were inhibited by oxygen at low levels (half-oxygen inhibition; ki,O2 ≈ 0.092 µM). The higher affinity of AOA for ammonia relative to AnAOB, suggests AOA/AnAOB cooperation is only possible where AOA do not outcompete AnAOB for ammonia. Using a biofilm model, we show that environments of ammonia/oxygen counter diffusion, such as stratified lakes, favors this cooperation.

Ammonia-oxidizing bacteria are the primary N2 O producers in an ammonia-oxidizing archaea dominated alkaline agricultural soil.

Most agricultural N2 O emissions are a consequence of microbial transformations of nitrogen (N) fertilizer, and mitigating increases in N2 O emission will depend on identifying microbial sources and variables influencing their activities. Here, using controlled microcosm and field studies, we found that synthetic N addition in any tested amount stimulated the production of N2 O from ammonia-oxidizing bacteria (AOB), but not archaea (AOA), from a bioenergy crop soil. The activities of these two populations were differentiated by N treatments, with abundance and activity of AOB increasing as nitrate and N2 O production increased. Moreover, as N2 O production increased, the isotopic composition of N2 O was consistent with an AOB source. Relative N2 O contributions by both populations were quantified using selective inhibitors and varying N availability. Complementary field analyses confirmed a positive correlation between N2 O flux and AOB abundance with N application. Collectively, our data indicate that AOB are the major N2 O producers, even with low N addition, and that better-metered N application, complemented by selective inhibitors, could reduce projected N2 O emissions from agricultural soils.

Influence of oxygen availability on the activities of ammonia-oxidizing archaea.

Recent studies point to the importance of oxygen (O2 ) in controlling the distribution and activity of marine ammonia-oxidizing archaea (AOA), one of the most abundant prokaryotes in the ocean. The AOA are associated with regions of low O2 tension in oceanic oxygen minimum zones (OMZs), and O2 availability is suggested to influence their production of the ozone-depleting greenhouse gas nitrous oxide (N2 O). We show that marine AOA available in pure culture sustain high ammonia oxidation activity at low µM O2 concentrations, characteristic of suboxic regions of OMZs (<10 µM O2 ), and that atmospheric concentrations of O2 may inhibit the growth of some environmental populations. We quantify the increasing N2 O production by marine AOA with decreasing O2 tensions, consistent with the plausibility of an AOA contribution to the accumulation of N2 O at the oxic-anoxic redox boundaries of OMZs. Variable sensitivity to peroxide also suggests that endogenous or exogenous reactive oxygen species are of importance in determining the environmental distribution of some populations.

Agricultural land usage transforms nitrifier population ecology.

Application of nitrogen fertilizer has altered terrestrial ecosystems. Ammonia is nitrified by ammonia and nitrite-oxidizing microorganisms, converting ammonia to highly mobile nitrate, contributing to the loss of nitrogen, soil nutrients and production of detrimental nitrogen oxides. Mitigating these costs is of critical importance to a growing bioenergy industry. To resolve the impact of management on nitrifying populations, amplicon sequencing of markers associated with ammonia and nitrite-oxidizing taxa (ammonia monooxygenase-amoA, nitrite oxidoreductase-nxrB, respectively) was conducted from long-term managed and nearby native soils in Eastern Washington, USA. Native nitrifier population structure was altered profoundly by management. The native ammonia-oxidizing archaeal community (comprised primarily by Nitrososphaera sister subclusters 1.1 and 2) was displaced by populations of Nitrosopumilus, Nitrosotalea and different assemblages of Nitrososphaera (subcluster 1.1, and unassociated lineages of Nitrososphaera). A displacement of ammonia-oxidizing bacterial taxa was associated with management, with native groups of Nitrosospira (cluster 2 related, cluster 3A.2) displaced by Nitrosospira clusters 8B and 3A.1. A shift in nitrite-oxidizing bacteria (NOB) was correlated with management, but distribution patterns could not be linked exclusively to management. Dominant nxrB sequences displayed only distant relationships to other NOB isolates and environmental clones.

Evaluation of revised polymerase chain reaction primers for more inclusive quantification of ammonia-oxidizing archaea and bacteria.

Ammonia-oxidizing archaea (AOA) and bacteria (AOB) fill key roles in the nitrogen cycle. Thus, well-vetted methods for characterizing their distribution are essential for framing studies of their significance in natural and managed systems. Quantification of the gene coding for one subunit of the ammonia monooxygenase (amoA) by polymerase chain reaction is frequently employed to enumerate the two groups. However, variable amplification of sequence variants comprising this conserved genetic marker for ammonia oxidizers potentially compromises within- and between-system comparisons. We compared the performance of newly designed non-degenerate quantitative polymerase chain reaction primer sets to existing primer sets commonly used to quantify the amoA of AOA and AOB using a collection of plasmids and soil DNA samples. The new AOA primer set provided improved quantification of model mixtures of different amoA sequence variants and increased detection of amoA in DNA recovered from soils. Although both primer sets for the AOB provided similar results for many comparisons, the new primers demonstrated increased detection in environmental application. Thus, the new primer sets should provide a useful complement to primers now commonly used to characterize the environmental distribution of AOA and AOB.

Influence of edaphic and management factors on the diversity and abundance of ammonia-oxidizing thaumarchaeota and bacteria in soils of bioenergy crop cultivars.

Ammonia-oxidizing thaumarcheota (AOA) and ammonia-oxidizing bacteria (AOB) differentially influence soil and atmospheric chemistry, but soil properties that control their distributions are poorly understood. In this study, the ammonia monooxygenase gene (amoA) was used to identify and quantify presumptive AOA and AOB and relate their distributions to soil properties in two experimental fields planted with different varieties of switchgrass (Panicum virgatum), a potential bioenergy feedstock. Differences in ammonia oxidizer diversity were associated primarily with soil properties of the two field sites, with pH displaying significant correlations with both AOA and AOB population structure. Percent nitrogen (%N), carbon to nitrogen ratios (C : N), and pH were also correlated with shifts nitrifier population structure. Nitrosotalea-like and Nitrosospira cluster II populations were more highly represented in acidic soils, whereas populations affiliated with Nitrososphaera and Nitrosospira cluster 3A.1 were relatively more abundant in alkaline soils. AOA were the dominant functional group in all plots based on quantitative polymerase chain reaction and high-throughput sequencing analyses. These data suggest that AOA contribute significantly to nitrification rates in carbon and nitrogen rich soils influenced by perennial grasses.

Disrupting mycorrhizal mutualisms: a potential mechanism by which exotic tamarisk outcompetes native cottonwoods.

The disruption of mutualisms between plants and mycorrhizal fungi is a potentially powerful mechanism by which invasives can negatively impact native species, yet our understanding of this mechanism's role in exotic species invasion is still in its infancy. Here, we provide several lines of evidence indicating that invasive tamarisk (Tamarix sp.) negatively affects native cottonwoods (Populus fremontii) by disrupting their associations with arbuscular mycorrhizal (AM) and ectomycorrhizal (EM) fungi. At a field site in the early stages of tamarisk invasion, cottonwoods with tamarisk neighbors had reduced EM colonization and altered EM fungal community composition relative to cottonwoods with native neighbors, leading to reductions in EM propagule abundance in the soil beneath tamarisk. Similarly, AM colonization of cottonwoods was reduced with a tamarisk neighbor, but there were no significant changes in AM fungal spore communities or propagule abundance. Root colonization by nonmycorrhizal fungi, including potential pathogens, was higher in cottonwoods with tamarisk neighbors. A greenhouse experiment in which AM and EM inoculation and plant neighbor were manipulated in a fully factorial design showed that cottonwoods benefited from mycorrhizas, especially EM, in terms of shoot biomass when grown with a conspecific, but shoot biomass was similar to that of nonmycorrhizal controls when cottonwoods were grown with a tamarisk neighbor. These results are partially explained by a reduction in EM but not AM colonization of cottonwoods by a tamarisk neighbor. Tamarisk neighbors negatively affected cottonwood specific leaf area, but not chlorophyll content, in the field. To pinpoint a mechanism for these changes, we measured soil chemistry in the field and the growth response of an EM fungus (Hebeloma crustuliniforme) to salt-amended media in the laboratory. Tamarisk increased both NO3- concentrations and electrical conductivity 2.5-fold beneath neighboring cottonwoods in the field. Salt-amended media did not affect the growth of H. crustuliniforme. Our findings demonstrate that a nonnative species, even in the early stages of invasion, can negatively affect a native species by disrupting its mycorrhizal symbioses. Some of these changes in mycorrhizal fungal communities may remain as legacy effects of invasives, even after their removal, and should be considered in management and restoration efforts.