Nitrous oxide (N
2O) emissions from grazed pastures are a product of microbial transformations of nitrogen and the prevailing view is that these only occur in the soil. Here we show this is not the case. We have found ammonia-oxidising bacteria (AOB) are present on plant leaves where they produce N
2O just as in soil. AOB (
Nitrosospira sp. predominantly) on the pasture grass
Lolium perenne converted 0.02–0.42% (mean 0.12%) of the oxidised ammonia to N
2O. As we have found AOB to be ubiquitous on grasses sampled from urine patches, we propose a ‘plant'' source of N
2O may be a feature of grazed grassland.In terms of climate forcing, nitrous oxide (N
2O) is the third most important greenhouse gas (
Blunden and Arndt, 2013). Agriculture is the largest source of anthropogenic N
2O (
Reay et al., 2012) with about 20% of agricultural emissions coming from grassland grazed by animals (
Oenema et al., 2005).Grazed grassland is a major source of N
2O because grazers harvest nitrogen (N) from plants across a wide area but recycle it back onto the pasture, largely as urine, in patches of very high N concentration. The N in urine patches is often in excess of what can be used by plants resulting in losses through leaching as nitrate, as N
2O and through volatilisation as ammonia (NH
3) creating a high NH
3 environment in the soil and plant canopy; an important point that we will return to later. The established wisdom is that N
2O is generated exclusively by soil-based microbes such as ammonia-oxidising bacteria (AOB). This soil biology is represented in models designed to simulate N
2O emissions and the soil is a target for mitigation strategies such as the use of nitrification inhibitors.We have previously shown that pasture plants can emit N
2O largely through acting as a conduit for emissions generated in the soil, which are themselves controlled to some degree by the plant (
Bowatte et al., 2014). In this case the origin of the emission is still the soil microbes. However, AOB have been found on the leaves of plants, for example, Norway spruce (
Papen et al., 2002;
Teuber et al., 2007) and weeds in rice paddies (
Bowatte et al., 2006), prompting us to ask whether AOB might be present on the leaves of pasture species and contribute to N
2O emissions as they do in soil.We looked for AOB on plants in situations where NH
3 concentrations were likely to be high, choosing plants from urine patches in grazed pastures and plants from pastures surrounding a urea fertiliser manufacturing plant. DNA was extracted from the leaves (including both the surface and apoplast) and the presence of AOB tested using PCR. AOB were present in all the species we examined—the grasses
Lolium perenne,
Dactylis glomerata,
Anthoxanthum odoratum,
Poa pratensis,
Bromus wildenowii and legumes
Trifolium repens and
T. subterraneum.To measure whether leaf AOB produce N
2O, we used intact plants of ryegrass (
L. perenne) lifted as cores from a paddock that had been recently grazed by adult sheep. The cores were installed in a chamber system designed to allow sampling of above- and belowground environments separately (
Bowatte et al., 2014). N
2O emissions were measured from untreated (control) plants and from plants where NH
3 was added to the aboveground chamber and leaves were either untreated or sterilised by wiping twice with paper towels soaked in 1% hypoclorite (
Sturz et al., 1997) and then with sterile water. We tested for the presence and abundance of AOB on the leaves by extracting DNA and using PCR and real-time PCR targeting the ammonia monoxygenase A (
amoA) gene, which is characteristic of AOB. AOB identity was established using cloning and DNA sequencing. Further details of these experiments can be found in the
Supplementary Information.The addition of NH
3 to untreated plants significantly stimulated N
2O emissions (
P<0.001) compared with the controls; by contrast, the plants with sterilised leaves produced significantly less N
2O than controls (
P<0.001) even with NH
3 added () providing strong evidence for emissions being associated with bacteria on the leaves. Control plants did emit N
2O suggesting there was either sufficient NH
3 available for bacterially generated emissions and/or other plant-based mechanisms were involved (
Bowatte et al., 2014).
Open in a separate windowEffect of an elevated NH
3 atmosphere and surface sterilisation of leaves on leaf N
2O emissions measured over 1-h periods on three occasions during the day. Values are means (s.e.m.), where
n=7.The major AOB species identified was
Nitrosospira strain III7 that has been previously shown to produce N
2O (
Jiang and Bakken, 1999). We measured 10
9 AOB cells per m
2 ryegrass leaf, assuming a specific leaf area of 250 cm
2 g
−1 leaf.The rate of production of N
2O (0.1–0.17 mg N
2O-N per m
2 leaf area per hour) can be translated to a field situation using the leaf area index (LAI)—1 m
2 leaf per m
2 ground would be an LAI of 1. LAI in a pasture can vary from <1 to >6 depending on the management (for example,
Orr et al., 1988). At LAI of 1, the AOB leaf emission rate would equate to a N
2O emission rate of about 0.1–0.3 mg N
2O-N per m
2 ground per hour. By comparison, the emission rates measured after dairy cattle urine (650 kg N ha
−1) was applied to freely and poorly drained soil were 0.024–1.55 and 0.048–3.33 mg N
2O-N per m
2 ground per hour, respectively (
Li and Kelliher, 2005).The fraction of the NH
3 that was converted to N
2O by the leaf AOB was 0.02–0.42% (mean 0.12%). The mean value is close to that measured for
Nitrosospira strains including strain III7 isolated from acidic, loamy and sandy soils where values ranged from 0.07 to 0.10% (
Jiang and Bakken, 1999). This is good evidence that the AOB on leaves have the capacity to produce N
2O at the same rate as AOB in soils. We do not suggest that leaf AOB will produce as much N
2O as soil microbes; however, because leaf AOB have access to a source of substrate—volatilised NH
3—that is unavailable to soil microbes and may constitute 26% (
Laubach et al., 2013) to 40% (
Carran et al., 1982) of the N deposited in the urine, N
2O emissions from these aboveground AOB are additional to soil emissions. Further research is required to identify the situations in which leaf AOB contribute to total emissions and to quantify this contribution.
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