Poster77: Biological nitrification inhibition: a dual benefit for agriculture and the environment
Biological Nitrification Inhibition (BNI):
a dual benefit for agriculture and the environment
D. E. Moreta1,3, M. P. Hurtado1, A. F. Salcedo1, L. Chávez1, M. Rondón1,M. C. Duque1,
G. V. Subbarao2, O. Ito2, J. Miles1, C. E. Lascano1; I. M. Rao1, & M. Ishitani1.
1International Center for Tropical Agriculture (CIAT) A.A. 6713 Cali, Colombia. 2Japan International Research Center for Agricultura1 Sciences (JIRCAS) 1-1 Ohwashi, Tsukuba, lbaraki
305-8686, Japan. 3Universidad del Valle.Departamento de Biología AA 25360. Cali, Colombia.
BNI in the plant-soil system
Nitrogen Cycle in Soil Significance of BNI in agricultural systems
Brachiaria humidicola: a case study
Nearly 70% of applied nitrogen fertilizers is lost in the agroecosystems.
The losses are estimated to be around US$ 16.4 billions annually from
cereal production systems alone across the world .
Nitrification process associated with emissions of N2O, NO, and N2, of
which, N2O has a greenhouse gas effect of 300 times higher than CO2.
Soil NO3- is highly prone to leaching and runoff losses.
Soil microorganisms convert NH4+ to nitrite (NO2-) and then to The tropical forage grass Brachiaria humidicola is well adapted BNI can improve sustainability of agroecosystems
nitrate (NO3-) through a process called nitrification. Oxidation of to acid soils of humid and sub-humid tropics; particularly to low- through its contribution to improved nitrogen use
NH4+ to NO2- is mediated by ammonia-oxidizing microorganisms fertility soils of South America. Root exudates of B. humidicola
through the ammonia-monooxigenase (amoA) enzyme pathway. inhibit the nitrification process. The inhibitory compound(s) in efficiency (NUE)
NO2- is then converted to nitrate (NO3-) and subsequently the root exudates specifically block the amoA enzymatic
denitrified to gaseous nitrogen. pathway of ammonia-oxidizing microorganisms. This
inhibitory effect is known as BNI.
To characterize the BNI phenomenon in tropical forages and rice in the plant-soil system. EVIDENCE OF HIGH BNI IN TROPICAL FORAGES
To identify genetic components responsible for BNI activity for improved NUE in crops.
Bioluminescence assay revealed the highest BNI activity in B. humidicola CIAT 16888 compared
METHODOLOGY to B. humidicola CIAT 679 and P. maximum, which confirms previous reports (Subbarao et al., 2006).
Bioluminiscence assay to detect nitrification inhibition in root exudates: This assay uses an Soil analysis revealed the highest levels of nitrate in the plots of soybean and the bare soil relative to
ammonia-oxidizing bacteria (Nitrosomonas europaea) transformed with the pHLUX20 plasmid that contains the that of B. humidicola. This indicated low rate of nitrification for B. humidicola due to high BNI activity
lux gene (Iizumi et al, 1998). As transformed Nitrosomonas emits luminescence, inhibitory effect of root exudates (Fig. 2C).
of crop species on its metabolic activity can be detected through bioluminescence (Fig.1).
Further confirmation through Real-Time PCR: higher gene copy number g-1 dried soil in Brachiaria
humidicola by exhibiting a marked reduction of AOB and AOA amoA gene copy number than that of
A bare soil and soybean (Fig. 2A and 2B).
Copies/g dry soil of AOBamoA gene
C o p y n u m b er
(m g/k g)
N -N O 3
B B 20 b
0.0E+00 B B
Bare soil Soybean Panicum maximum Hybrid mulato B. humidicola 679 B. humidicola 16888 10
d d c
Figure 1. Bioluminescence assay: A. Physical map of pHLUX20 used to transform N. europaea. Soil DNA 0
Hybrid Mulato Bare soil Bh 679 Bh 16888 P.maximum Soybean
B. Brachiaria humidicola plants growing in nutrient solution at greenhouse to obtain root exudates.
Copies/g dry soil of AOA amoA gene
B Figure 2. Influence of tropical pasture grass
BNI assays at the field: 1.0E+08 cultivation on soil microorganism populations at 1
Location: CIAT Palmira (3o 30’ N, 76o 21’ W), Altitude: 965 masl; Vertisol (Typic Pellustert), pH 7.4, annual mean
day after ammonium-sulfate fertilization by
rainfall: 1000 mm, annual mean temperature:26º C. A
estimating copy number of A. AOB amoA gene and
Experimental design: RBD, 3 Replications, 10 m X 10 m plot size, 5 crop species and bare soil as control. 2.0E+07 B B
B. AOA amoA gene. C. Nitrate (NO3-) levels in soil
0.0E+00 1 day after fertilization. Gene copy number was
N-Fertilizer application: A localized application of liquid ammonium sulfate in 2 subplots (1m X 1m) in each plot. Bare soil Soybean Panicum Hybrid mulato B. humidicola B. humidicola expressed as copy number per g of dried soil and
Soil sampling: soil samples collected for quantification of ammonia-oxidizing microorganism genes by Real-Time maximum 679 16888
obtained through absolute quantification by using
PCR. Sampling of soil coincided with 4th and 5th harvest of foliage of crop species to simulate grazing in grasslands. Real-Time PCR. Values are means + SE. Values
with different letters are significantly different.
Crop species BNI activity
Brachiaria humidicola CIAT 16888 High RICE SHOWING POTENTIAL FOR BNI ACTIVITY
Brachiaria humidicola CIAT 679 High Sampling times:
Panicum maximum Low Utilization of diverse rice germplasm for BNI screening
1 day before fertilization
Brachiaria hybrid cv. Mulato Intermediate 3 biological replications
Soybean (nitrification enhancer) Not detected 1 day after fertilization ~100 rice genotypes are being screened for BNI activity representing the biodiversity of rice worldwide.
30 days after fertilization
Bare soil (no plants) As control Elucidation of mode of BNI activity using contrasting rice genotypes
Preliminarily results have revealed high BNI activity in a rice genotype (line BNI 32). This finding requires
Collecting soil samples
B. humidicola Bare soil further confirmation (Fig. 3).
3 subplot representative
Figure 3. Preliminary evidence
soil samples from
of BNI activity of the line BNI
32, an upland rice genotype, by
~10 cm soil depth measuring A) nitrite formation
(mg/kg) in rhizosphere soil, and
B) Nitrification rate (mg/kg
Soil bacterial gene quantification by Real-Time PCR: DNA from soil samples were isolated using the soil/day) in soil.
FastDNA® SPIN for soil kit (MP Biomedicals) and quantified by fluorescence with the PicoGreen® dsDNA
quantification reagent (Molecular Probes). Copy number of four target genes; i.e. Bacteria Small-Subunit (SSU) A B
rRNA gene, ammonia-oxidizing bacteria (AOB) amoA gene, Archaea SSU rRNA, and ammonia-oxidizing
archaea (AOA) amoA gene were quantified through Real-Time PCR using specific primer combinations. All
target genes were quantified with the SYBR® Green I as a fluorescent dye. Real-Time PCR reactions were PERSPECTIVES
performed in triplicate using a Engine OPTICONTM2 Continuous Fluorescence Detector thermocycler (MJ
Research) and analyzed with the MJ OPTICON MonitorTM Analysis Software version 3 (BIO-RAD). Raw data Identification of compounds responsible for BNI in rice root exudates.
(gene copy number reaction-1) were corrected for soil gravimetric moisture content and then expressed as gene Identification of genetic components responsible for BNI trait in rice towards crop
copy numbers g-1 dried soil by employing an algebraic standard operating procedure. improvement.
Soil analysis: Soil nitrate (NO3-) and ammonium (NH4 +) were measured by ultraviolet visible spectroscopy ACKNOWLEDGEMENTS
technique at 410 nm and 667 nm, respectively.
BNI research in rice is being supported by Bioversity International (Vavilov-Frankel Fellowship, 2009).
Iizumi, T., et al. 1998. A Bioluminescence Assay Using Nitrosomonas europaea for Rapidand Sensitive Detection of Nitrification Inhibitors Applied and Environmental Microbiology Vol. 64, No. 10 p. 3656–3662
Leininger, S., et al. 2006. Archaea predominate among ammonia-oxidizing prokaryotes in soils Archaea predominate among ammonia-oxidizing prokaryotes in soils Nature Vol 442 August 2006|
Subbarao G. V., et al. 2006. A bioluminescence assay to detect nitrification inhibitors released from plant roots: a case study with Brachiaria Humidicola Plant Soil
Okano, Y., et al 2004. Application of Real-Time PCR To Study Effects of Ammonium onPopulation Size of Ammonia-Oxidizing Bacteria in Soil Applied and Environmental Microbiology Vol. 70, No. 2 p. 1008-1016