1. Effects of Nitrogen Fertilization on the Abundance
of Ammonia Oxidizing Archaea and Bacteria
in an Agricultural Soil
A.E. Prado¹, L.G. Whyte¹, J.K. Whalen¹, M.R. Chénier² ³
¹Department of Natural Resource Sciences,²Department of Food Science, ³Department of Animal Science, McGill University
adrian.prado@mail.mcgill.ca
amo
,
• Ammonia oxidation is the rate-limiting step of the nitrogen cycle.
• It is a process mediated by ammonia oxidizing bacteria (AOB) and
archaea (AOA).
• In agricultural systems AOA and AOB compete with crops for ammonia.
• Ammonium and urea-based fertilizers are common and are converted
into ammonia, which is metabolized by AOA and AOB.
• Ammonia remains in soils, while the products of ammonia oxidation do
not and this can cause important environmental, economic and health
issues.
• The enzyme responsible for ammonia oxidation is ammonia mono-
oxygenase, encoded by the operon and present in AOA and AOB.
• Quantify the effects of nitrogen fertilization on the abundance of
ammonia oxidizers
• Assess the effects of crop growth stage on the abundance of ammonia
oxidizers
DNA Extraction
Kit
SYBR Green
QPCR
454
Pyrosequencing
Soil
Figure 1: Simplified Molecular
Methodology
Table 1: Primers used in this Study
• Four nitrogen fertilizer treatments were applied to an experimental
canola field in Ottawa, Ontario: 0, 50, 100 and 150 kg N/ha.
• Soil samples were taken at three key growth stages of the plants:
rosette formation, flowering and pod formation.
• The experiment was conducted in triplicate.
• Pyrosequencing has been performed by Génome Québec and bio-
informatics analysis is underway to assess community structure changes.
This work was primarily funded by FRQNT through a grant (Projet de recherche en équipe) held by Martin R. Chénier.
Additional support was provided to Martin R. Chénier by NSERC through a Discovery Grant and by the Office of the Vice-
Principal - Research andInternational Relations (McGill).
0
1.00x10⁶
2.00x10⁶
3.00x10⁶
4.00x10⁶
5.00x10⁶
6.00x10⁶
7.00x10⁶
Rosette (08/06/2012) Flowering (29/06/2012) Pod Formation (13/07/2012)
Copies/mgofDrySoil
Figure 3: Beta-Proteobacterial (AOB) amoA Copies
0 kg N/ha
50 kg N/ha
100 kg N/ha
150 kg N/ha
0
1.00x10⁶
2.00x10⁶
3.00x10⁶
4.00x10⁶
5.00x10⁶
6.00x10⁶
7.00x10⁶
Rosette (08/06/2012) Flowering (29/06/2012) Pod Formation (13/07/2012)
Copies/mgofDrySoil
Figure 2: Thaumarchaeal (AOA) amoA Copies
0 kg N/ha
50 kg N/ha
100 kg N/ha
150 kg N/ha
Introduction
Objectives
Methods
Results
Discussion
Target Primer Pair QPCR 454
beta-proteobacterial amoA amoA1-F/amoA2-R X X
gamma-proteobacterial amoA amoA151F/amoA682R X
Thaumarchaeal amoA CrenamoA23F/Crenamo616R X X
16S Archaea and Bacteria 515 F/806R X X
Effect p-value Significance
Fertilizer Treatment 1.09x10 **
Plant Growth Stage 1.30x10 **
Interaction 2.89x10 *
Effect p-value Significance
Fertilizer Treatment 1.87x10 -
Plant Growth Stage 3.67x10 **
Interaction 5.78x10 **
• Extraction was performed with the
MOBio Powersoil DNA Isolation Kit.
• qPCR Standards were derived from
agarose gel purified PCR amplicons
subsequently serially diluted.
• All samples were diluted to 2 ng/µL.
• qPCR was used to quantify amoA
of each group and 16S.
• qPCR Efficiencies: 95-105%,
R²>0.99
-1
-40
-15
-68
-5
-2
• Regardless of the growing stage, AOA are more abundant than
AOB at lower N concentrations, but the opposite occurs at high N.
• AOB dominance under the high N treatments is more pronounced
early on in the development of the plant, when nutrients are more
abundant, but the trend reverses as the season progresses.
• In contrats, in the absence of N fertilizer, the abundance of AOA
is higher than that of AOB throughout the growing season.
• Nitrogen fertilization promotes the growth of AOB over AOA, while plant
development (and potential nutrient depletion) reverses the effect.
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Acknowledgements
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