This document summarizes current research on biological nitrification inhibition (BNI) being conducted at the Japan International Research Center for Agricultural Sciences (JIRCAS). It discusses BNI research collaborations with other institutions and lists JIRCAS colleagues contributing to the work. Key findings include the identification of brachialactone as the major nitrification inhibitor produced by Brachiaria humidicola roots and seasonal variation in its release. The document also examines genetic variability in BNI capacity between B. humidicola accessions, suggesting potential for traditional breeding to improve this trait in forage grasses.

1. Current Status of BNI Research at JIRCAS
GV Subbarao
JIRCAS, Japan
A Collaborative effort with CIAT, ICRISAT and CIMMYT
Collaborators
CIAT
CIMMYT
ICRISAT
Tottori University
Scottish Crops Research Institute
Colleagues contributed from JIRCAS
1. T. Ando
2. K. Nakahara
3. T. Yoshihashi
4. T. Watanabe
5. T. Ishikawa
6. Y. Yamanaka
7. H. Y. Wang (PDF)
8. S. Gopalakrishnan (PDF)
9. Stuart Pearse (PDF)
10. A.K.M. Hussain (PDF)
11. Yiyong Zhu (PDF)
12. Zhu Yiyong (PDF)
13. T. Tsehaye (PDF)

2. Nearly 70% of the N fertilizer applied is lost to the environment
Amounts to a direct annual economic loss of
US$ 90 billion*
[*based on - a) world annual N fertilizer production is 150 million Mg; b) 0.45 US$ kg-1 urea]
Nitrogen fertilizer consumed in 1930s - < 1.0 Tg (million metric tons)
Nitrogen fertilizer consumed in 1960s – 10 Tg
Nitrogen fertilizer consumption worldwide in 2010 – 150 Tg (million metric tons)
Energy cost of nitrogen fertilizer – 1.8 to 2 L diesel oil per kg N fertilizer
To produce 150 million metric tons of Nitrogen fertilizer requires
1.70 billion barrels of diesel oil (energy equivalent)
Nitrogen fertilizers – Some facts

3. Year
1950 1960 1970 1980 1990 2000 2010 2020
Nitrogenefficiencyincerealproduction
(megatonnescerealgrain/megatonnsfertilizerapplied)
20
30
40
50
60
70
80
Trends in N-fertilization efficiency in cereal production
(annual global cereal production divided by annual global application of N-fertilizer) (Source: FAO 2012)
Global food production has tripled during this period, but N-fertilizer
applications have increased 10-fold (Tilman et al., 2001)

4. Why NUE is <30% in most agricultural systems?
Nitrification and denitrification processes associated with uncontrolled
rapid nitrification are largely responsible for the massive N leakage
(>70% of the N fertilizers) and for the low-NUE

5. Nitrogen Cycle in Typical Agricultural Systems
Soil
OM
Organic N
NH4
+
Microbial
N
NO3
-
>95% of the total soil
inorganic N pool
Plant N uptake &
Assimilation
Mineralization
Nitrification
Inorganic
N
Crop
Residues
N
Fertilizer

6. Soil incubation period in days
0 10 20 30 40
Nitrification(%)
0
20
40
60
80
100
120
Intensively managed
Alfisols
Watersheds
Conservatively managed Alfisols
Alfisol fields at ICRISAT
WS HP
Nitrificationrate(gNO3
-g-1soild-1)
0
1
2
3
4
5
Alfisol fields at ICRISAT
WS HP
Nitrificationrate(gNO3
-g-1soild-1)
0
1
2
3
4
5
Conservatively managed
Watershed Alfisols
Intensively managed
High-precision Alfisols
Agricultural intensification led to acceleration
of nitrification in intensively-managed
production systems

7. How to achieve low-nitrifying agricultural soils?
Switch to low-nitrifying agricultural systems

8. Ammonium
(NH4
+)
Nitrite
(NO2
-)
 Leaching
Nitrate
(NO3
-)
 N2O, NO, N2
 Greenhouse gases
 Global warming
 Nitrification
OM
mineralization  Denitrification
Ammonia-oxidizing Bacteria Nitrite-oxidizing Bacteria
Biological Nitrification Inhibition (BNI)
Brachiaria spp.
root-produced
nitrification
inhibitors
Microbial
Immobilization
of NH4
+
Low-Nitrifying Natural Ecosystems High-Nitrifying Modern Agricultural Systems
BL
BL
BL
BL
BL
BL
BL
BL
BL
BL
BL
NFertilizer
BNI Function and its potential impacts to N-cycling

9. How to detect and quantify nitrification inhibitors ?
pHLUX20
9763 bp
(Bg/II/
BamHl)
kat
Trrn
Phao
luxAB
PstI
(BamHI/Bg/II) PstI
PstI
BamHI
Physical map of pHLUX20
(source: Iizumi et al. 1998)
OM
IM
NH2OH + H2O NO2
- + 5H+ + 4e-
NH3 + O2
HAO
c554 c554
UQ
UQH2
NAD(P)H + H+ NAD(P)+
FMNH2FMN
H2O
hv RCOOH
RCHO
O2
Luciferase
NAD(P)+
reductase
Cytaa3
oxidase
NAD(P)H-FMN
AMO
oxidoreductase
Hypothetical model of interaction between the
electron transfer pathways and the luciferase
reaction in N. europaea (source Iizumi et al. 1998)
BNI activity is expressd in
‘ATU’
Inhibitory effect from 0.28
M AT is defined as one
ATU

10. Pasture grasses
0 1 2 3 4 5 6 7
BNI-activityreleasedfromroots
(ATUg
-1
rootdrywt.d
-1
)
0
2
4
6
8
10
12
14
16
18
1. B. humidicola
2. M. minutiflora
3. P. maximum
4. L. perenne
5. A. gayanus
6. B. brizantha
BNI capacity of pastures
JIRCAS-CIAT partnership

11. Plants release two categories of BNIs
Hydrophobic Hydrophilic
BNI Activity
Mostly confined to
Rhizosphere
May move out of
Rhizosphere
Plant -root
produced
nitrification
inhibitors
BL
BL
BL
BL
BL
BL
BL
BL
BL
BL
BL
BL
BL
BLBL
BL
BL
BL
BL

12. Plant Species
BH Sorghum Wheat
BNIactiviity(%oftotalBNIactivity)
0
20
40
60
80
100
Hydrophobic-BNI
Hydrophilic-BNI
Relative importance of hydrophobic- and
hydrophilic- BNI activity in three plant species
at 8 d old plants
40 d old plants
8 d old plants

13. BNI activity added to the soil (AT g-1 soil)
0 5 10 15 20 25
NO3concentrationinsoil(ppm)
0
50
100
150
200
250
Threshold
Releases about 200 to 400 ATU hydrophilic BNI d-1
BNIs provide stable inhibitory effect on
soil nitrification
55 d soil incubation

14. 14
A bioassay-guided purification of BNI activity led to isolation of
Brachialactone,
identified as the major nitrification inhibitor
released from the roots of B. humidicola.
Atricyclic terpenoid with a unique
5-8-5 ring system and a g-lactone ring
Similar 5-8-5 ringsystem
Fusicoccins are produced
in some fungi
(Geranylgeranyl diphosphate)
Patented by JIRCAS
GGDPis a precursorin thebiosynthesisof
terpenoids;also thisis theprecursorfor thesynthesis
ofcarotenoids, gibberllinsand chlorophylls in plants
11
Subbarao G V et al. PNAS 2009;106:17302-17307
©2009 by National Academy of Sciences

15. 0.0 2.5 5.0 7.5 10.0 12.5 15.0 min
0
5000
10000
15000
20000
25000
30000
35000
40000
45000
uV
sample: brachialactone standard (mixture of a and b), 75 μg
column: TSK gel super ODS (4.6 x 100 mm)
mobile phase: water (A) – acetonitrile (B)
flow rate: 1.0 ml/min
gradient program: 23% - 43%B (10 min), 43% - 48%B (8 min)
Time (min)
Detectorresponse
Brachialactone b
Purified Brachialactone HPLC chromatogram

16. GC-MS-SIM based brachialactone quantification
16
Progesterone (IS:1 ppm)
Brachialactone (48 ppm)
19.78min
Identification: m/z 334
Quantification: m/z 137
18.65min
Identification: m/z 314
Quantification: m/z 314
Quantification & identification was achieved.
Brachialactone showed 2 peaks,
which might be caused by keto-enol
tautomerism.
‘Keto’form ‘Enol’form

17. GC-MS-SIM based analytical methodology can have major
implications to genetic improvement efforts directed at
brachialactone-trait into root systems of Brachiaria sp.
Brachialactone is detected in root tissues and quantification using GC-
MS-SIM analysis could be a possibility in future
Preliminary results suggest brachialactone concentration in root tissues
can be as high as 0.27  0.01% (dry weight basis)
Brachialactone levels in root tissues could be up to 10 times higher
than in root exudates (i.e. about 10% of brachialactone in the root
tissues may be released per day from exudation)
GC-MS-SIM analysis improves the detection thresholds for
brachialatone levels in the samples and may give better quantification in
root tissues and root exudates.

18. Brachialactone release is highly influenced by
growing season
Spring season in Japan appears to have a major influence on brachialactone release in B. humidicola
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peakarea
mAU*sec.
date
Annualfluctuationrootexdate
standardBH
highBNIBH
2011 20132012
We need to understand whether these seasonal influence on brachialactone release from
root due to production in root tissues or only release from roots is influenced?

19. Brachialactone’s mode of inhibitory action on
Nitrosomonas
Compound
Concentration in the
in vitro assay, mM AMO pathway HAO pathway
Crude-root exudate
(methanol extract) 63.4 + 0.8 63.8 + 0.8
Brachialactone 5.0 59.7 + 0.9 37.7 + 0.9
Nitrapyrin 3.0 82.3 + 1.5 8.1 + 1.2
Inhibition (%)
Outer Membrane
Inner Membrane
Periplasm
Nitrosomonas

20. Regulating factors for the Release of BNIs from roots

21. BNI synthesis and release from roots requires presence of NH4
+
N treatment (NO3-N vs NH4-N grown plants)
NO3-grown NH4-grown
BNIactivityoftheroottissue(ATunitsg-1rootdrywt)
0
50
100
150
200
Root tissue from RE-water treatment
Root tissue from RE-NH4 treatment
Nitrogen treatment (i.e. NH4-N vs. NO3) of the plants
NO3-grown NH4-grown
TotalBNIactivityreleasedduring10dperiod(ATunits)
0
200
400
600
800
1000
RE-collected using distilled water
RE-collected using 1 NH4Cl (1 mM)

22. Functional link between NH4
+-uptake and BNI release
A hypothesis
NH4
+
Cytoplasm
pH >7
NH4
+ NH4
+
H+
H+
ATP
ADP + Pi
BNIn-
BNIn-
BNI
Glutamine + H+
glutamate

23. Is there potential for genetic
improvement of BNI capacity in pastures?
Genetic variability is the primary requirement for genetic
improvement in trait/s of interest using traditional breeding

24. Is there a genetic variability for BNI capacity?
High-BNI and low-BNI genetic stocks in B. humidicola
B. humidicola
Accession
BNI released
ATU g-1 root dry wt. d-1
CIAT 26159 46.3
CIAT 26427 31.6
CIAT 26430 24.1
CIAT 679 17.5
CIAT 26438 6.5
CIAT 26149 7.1
CIAT 682 7.5
Panicum maximum 0.1
LSD (0.05) 6.0
Based on evaluation of
40 germplasm accessions
in B.humidicola
CIAT’s Collaboration
Note
11 sexuals from a total of
40 germplasm accessions
were evaluated for BNI
capacity; Most sexuals
evaluated have BNI
capacity similar to the
CIAT 679.
A bi-parental population using high-BNI (CIAT 16888) and low-BNI (CIAT 26146)
has been developed to identify genetic regions associated with BNI-function using a
mapping population derived from crosse between apomictic and sexual germplasm
accession of BH that differ in BNI-capacity – CIAT-JIRCAS ongoing collaboration

25. Date of Root exudate collection during Spring 2012
2nd March 3rd March 4th March 1st April
Brachialactonereleaseperplant
(peakarea)
0
2000
4000
6000
8000
10000
12000
CIAT 679
CIAT 26159
CIAT26159
CIAT679
Genetic differences in
Brachialactone release capacity
High-BNI genotype releases several times higher
brachialactone than standard cultivar
25

26. Parental lines of RIL population
PVK 801 296-B
BNIactivity/Sorgoleonereleaseperplant
0
10
20
30
40
50
BNI activity (ATU)
Sorgoleone (g)
Total BNI activity and sorgoleone levels in root-DCM wash after 8 d growth
in root boxes with hydroponic system
(based on 6 times evaluation of 20 seed lings each over a 6 month period)
Parental lines of RIL population characterization
JIRCAS-ICRISAT
partnership

27. HPLC chromatogram of purified sorgoleone
BNI activity detected only in this peak
NO BNI activity detected in any of these peaks
O
O
OH
O
Chemical structure of sorgoleone, Molecular Weight - 358
a P-benzoquinone exuded from sorghum roots
BNI activity released from
sorghum roots
Hydrophobic BNIs
Hydrophilic BNIs
Isolation of the major BNI constituent
of hydrophobic BNI activity
ED80 = 1.0 ppm
Adroplet of sorgoleone
exuding from root tip

28. 296B PVK 801
Sorgoleone-phenotyping system is now developed
JIRCAS-ICRISAT
partnership

29. Bi-Parental Sorghum RIL population (PVK 801 x 296B)
0 50 100 150 200
Sorgoleoneproduced(gplant-1)
0
10
20
30
40
50
PVK801
296B
RIL phenotyping for sorgoleone levels in root-DCM wash
JIRCAS-ICRISAT partnership

30. Introducing high-BNI capacity into wheat
Is it possible or feasible?
JIRCAS-CIMMYT
partnership

31. Plant species
0 1 2 3 4
BNIactivityreleasedfromroots
(ATUg-1rootdrywt.d-1)
0
5
10
15
20
25
30
35
NH4-N grown
NO3-N grown
Nobeoka Chinese Spring
L. racemosus
Releases about 150 to 200 AT units of
BNI da-1 under optimum conditions
Wild-wheat has high-BNI capacity
JIRCAS-CIMMYT
partnership

32. Leymus racemosus
2N=4X =28;
genome Ns NsXmXm
Triticum aestivum L.
cv. Chinese Spring
2N=6X =42;
genome AABBDD
F1 hybrid Triticum aestivum L.
cv. Chinese Spring
2N=6X =42;
genome AABBDD
BC1F1 hybrid
BC7F1 hybrid
Production of wheat-Leymus racemosus-addition lines
Two Lr#n L. racemosus chromosomes in wheat detected by florescence in
situ hybridization with probe of L. racemosus genomic DNA (green color)
3.9LSD (0.05)
4.97Lr-1-2DtA7Lr-1-2
6.47Lr-1-1DtA7Lr-1-1
6.65Lr-1DA5Lr-1
3.22Lr-1DA2Lr-1
3.7Lr-HDALr-H
4.1Lr-FDALr-F
5.5Lr-kDALr-k
6.4Lr-1DALr-1
13.0Lr-IDALr-I
13.5Lr-jDALr-j
24.6Lr-nDALr-n
BNI released
(ATU g-1 root dry
wt d-1)
L. racemosus
chromosome
introduced
Genetic Stock
3.9LSD (0.05)
4.97Lr-1-2DtA7Lr-1-2
6.47Lr-1-1DtA7Lr-1-1
6.65Lr-1DA5Lr-1
3.22Lr-1DA2Lr-1
3.7Lr-HDALr-H
4.1Lr-FDALr-F
5.5Lr-kDALr-k
6.4Lr-1DALr-1
13.0Lr-IDALr-I
13.5Lr-jDALr-j
24.6Lr-nDALr-n
BNI released
(ATU g-1 root dry
wt d-1)
L. racemosus
chromosome
introduced
Genetic Stock
BNI released from Chromosome-addition lines derived from
L. racemosus and cultivated wheat (Chinese Spring)
Can the high-BNI capacity of wild-wheat be
Transferred/Expressed in cultivated wheat?
Would this be the first step to develop low-nitrifying and low-N2O
emitting wheat production systems?
BA
JIRCAS-CIMMYT
partnership

33. Lr#nS.3BL
Lr#nS.7BL
Leymus chromosome ‘N’
The short-arm of the Leymus ‘N’ chromosome is translocated to
either 7B or 3B wheat chromosome (short-arm) for BNI evaluations
Short arm
long arm
centromere
Courtesy - Kishi
Courtesy - Kishi
JIRCAS-CIMMYT
partnership

34. Lr#n addition
Lr#nS.3BL
Wheat-Leymus genetic stocks
CS N - add N - sub-3A N - Tr-3B N - Tr-7B
BNIactivityreleasedfromroots
(ATUg-1rootdrywt.d-1)
0
100
200
300
400
500
RE-NH4
+
BNI activity release from roots in the presence of
NH4
+ in the collection solutions
Courtesy - Kishi
Courtesy - Kishi
BNI activity release is two-fold higher in Lr#N addition and
Lr#N translocation line (on 3B wheat chromosome)
compared to Chinese Spring
The above results strongly confirm that BNI-capacity in Leymus is
controlled by Lr#N and expressed in wheat background; further the
BNI-trait is controlled by short-arm of Lr#n chromosome and its
expression depends on the translocation position on wheat
JIRCAS-CIMMYT
partnership

35. Can the BNI function be effective to
control nitrification and nitrous oxide
emissions under field conditions?
JIRCAS-CIAT partnership

36. Roots of B. humidicola release a powerful
nitrification inhibitor
Brachialactone
Ammonium
(NH4
+)
Nitrite
(NO2
-)
Nitrate
(NO3
-)Ammonia-oxidizing Bacteria Nitrite-oxidizing Bacteria
BL
BL
BL BL
Microbial-N
Immobilization
Mineralization
By blocking the Nitrosomonas function, B.
humidicola facilitates NH4
+ to move into
mocrobial pool and to remain in the soil
system and act as a slow-releasing nitrogen
source for Brachiaria growth

37. Estimations for the BNIs release from B. humidicola
• Active root biomass in a long-term BH pasture being 1.5 Mg ha-1
•(Root mass up to 9.0 Mg ha-1 has been reported in BH pastures)
• BNI release rates can be 17 to 50 ATU g-1 root dry wt. d-1
• Estimated BNI activity release d-1 could be 2.6 x 106 to 7.5 x 106
ATU
(CIAT 679) (CIAT 26159)
•1 ATU being equal to 0.6 g of nitrapyrin
• This amounts to an inhibitory potential equivalent to the
application of 6.2 to 18 kg of nitrapyrin application ha-1 yr-1

38. 38
Soil ammonium oxidation rates (mg of NO2− N per kg of soil per day) in field plots
planted with tropical pasture grasses (differing in BNI capacity) and soybean
(lacking BNI capacity in roots) [over 3 years from establishment of pastures
(September 2004 to November 2007); for soybean, two planting seasons every year
and after six seasons of cultivation]
Brachiaria pastures suppressed soil ammonium oxidation
Subbarao G V et al. PNAS 2009;106:17302-17307
©2009 by National Academy of Sciences
JIRCAS-CIATpartnership
CIAT-Palmira field study 2004-2007
0
0.5
1
1.5
2
2.5
3
3.5
4
Control - Bare soil BH- 16888
ppmofnitrateproducedday-1
CIAT-Palmira field study 2013

39. 39
Cumulative N2O emissions (mg of N2O N per m2 per year) from field plots of tropical pasture grasses
(monitored monthly over a 3-year period, from September 2004 to November 2007)
Subbarao G V et al. PNAS 2009;106:17302-17307
©2009 by National Academy of Sciences
Brachiaria pastures suppressed N2O emissions from the field
Can BNI function in plants be exploited to develop low-N2O emitting systems then?
JIRCAS-CIATpartnership

40. BNI capacity of the species (ATU g-1 root dry wt. d-1)
0 10 20 30 40 50 60
CumulativeN2Oemission
(mgN2O-Nm2y-1)
0
100
200
300
400
500
Con
Soy
PM
BHM
BH-679
BH-16888
High BNI capacity leads to low-N2O
emitting systems?
A 3-year field study with soybean and pasture grasses with varying BNI capacities
Can we develop low-nitrifying and
low-N2O emitting pasture-production
systems through genetic exploitation
of BNI trait?
The new MAFF-BNI project (starts from 2014) will test this hypothesis further using
genetic stocks of B. humidicola with diverse BNI capacity in root systems
JIRCAS-CIATpartnership

41. Photo: J. W. Miles Exploitation of BNI function in BH for the sustainable agro-pastoral systems?
Characterization of residual effect of BNI from
B. humidicola pasture on maize productivity and
Nitrogen use efficiency
Ongoing
JIRCAS-CIAT partnership

42. How long the BNI-suppressive effect on nitrification persists?
Ongoing
JIRCAS-CIAT partnership
Land Management
0 1 2
Nitrificationrate
(mgNO2-Nkg-1soild-1)
0.00
0.02
0.04
0.06
0.08
0.10
0.12
Native savanna
BH
Cultivated fields
Maize
BH-BNI effect
Time in years
0 1 2 3 4 5 6
Ammoniumoxidationrateinsoil
(mgNO2kg
-1
soild
-1
)
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
Cultivated soils
control
BH-residual
scenario-4
BH-residual
scenario-3
BH-residual
scenario-2
BH-residual
scenario-1

43. Characterization of residual BNI impact on NUE in maize systems
An agro-pastoral systems perspective
Ongoing
JIRCAS-CIAT partnership

44. Maize crop established in a high-BNI field by clearing B. humidicola
Field site – Taluma, Iianos, Colombia
JIRCAS – CIAT collaborative study – CIAT field site at Llanos
Ongoing
JIRCAS-CIAT partnership

45. 120 kg N/ha 240 kg N/ha
B. humidicolafieldThe BH-BNI benefits on
Maize growth
Beneficial effects of BNI on subsequent maize crop
Land Management
0 1 2
Nitrificationrate
(mgNO2-Nkg-1soild-1)
0.00
0.02
0.04
0.06
0.08
0.10
0.12
Native savanna
BH
Cultivated fields
Maize
BNI-Field
Ongoing
JIRCAS-CIAT partnership

46. 120 kg N ha-1
Beneficial effects of BNI on subsequent maize crop
A healthy maize crop in BNI-field with 120 kg N application
Land Management
0 1 2
Nitrificationrate
(mgNO2-Nkg-1soild-1)
0.00
0.02
0.04
0.06
0.08
0.10
0.12
Native savanna
BH
Cultivated fields
Maize
JIRCAS – CIAT collaborative study – CIAT field site at Llanos
BNI-Field
Ongoing
JIRCAS-CIAT partnership

47. 120 kg N ha-1
Land Management
0 1 2
Nitrificationrate
(mgNO2-Nkg-1soild-1)
0.00
0.02
0.04
0.06
0.08
0.10
0.12
Native savanna
BH
Cultivated fields
Maize
JIRCAS – CIAT collaborative study – CIAT field site at Llanos
Non-BNI-Field
Beneficial effects of BNI on subsequent maize crop
A nitrogen-deficient maize crop in non-BNI-field with 120 kg N
application
Ongoing
JIRCAS-CIAT partnership

48. BNI-Field Non-BNI-Field
2012 Field study at Iianos, Colombia
Nitrogen fertilizer application (Kg ha-1
)
40 60 80 100 120 140 160 180 200 220 240 260
Maizegrainyield(tha
-1
)
0
1000
2000
3000
4000
5000
High nitrifying - cultivated fields
Low nitrifying - BH-BNI
Beneficial effects of BNI on subsequent maize grain yields
BNI is more effective on maize yields at low to moderate N applications but not high-N environments
BNI function is effective in improving NUE only under low- to moderate-N environments
and not at high-N environments
BNI-field
Non-BNI-field
Ongoing
JIRCAS-CIAT partnership

49. Maize plant tissues from various land-use systems
Ear Shoot Root
15N/14Nratioinplanttissues
4.5
5.0
5.5
6.0
6.5
BH-BNI
cont.Maize
Native savannah
Beneficial effects of BNI on N recovery by Maize
BNI is effective in improving N recovery by maize in the field (from 15N studies)
BNI-Field
Non-BNI-Field
Ongoing
JIRCAS-CIAT partnership

50. Land use treatments on Maize
BH-BNI cont.Maize Native savannah
15N/14Nratioinsoils(0-60cmsdepth)
0.35
0.40
0.45
0.50
0.55
Beneficial effects of BNI on soil-N retention
BNI is effective in improving soil-N retention after maize harvest (from 15N studies)
BNI-Field
Non-BNI-Field
Ongoing
JIRCAS-CIAT partnership

51. CONCLUDING REMARKS

52. 175 Tg N
N-Fertilizer inputs
into Agriculture
53.5 Tg N
Plant protein from
Agriculture
3.5 Tg N
Animalprotein
from Livestock
0.27Tg N
Human system
N-retention
123.5 Tg N
LOST
(70%)
FromAgriculture
48.0 Tg N
LOST
(90%)
From Livestock
5.0 Tg N
LOST
(95%)
From Municipal
Sewage systems
N-Fertilizer inputs
into Agriculture
Plant
protein-N
Animal
protein-N
Human-N
Nitrogen flow in Human-centric Ecosystems
Annual

53. Nitrogen pollution epidemic in China
Nitrification facilitates movement of N from agricultural soils to water-bodies (ground
water, freshwater lakes, rivers and to oceans) and cause algal blooms
Second Green Revolution?

54. NH4
+
NO3
-
Plant
uptake
Soil-
microbial
uptake
Nitrification
SOM
Mineralization
N-Fertilizers
Microbial-N
Immobilization
Plant litter
and
Root exudates
Nitrification opens several pathways in N-cycle for
fertilizer-N to escape into the larger Environment

55. A fundamental shift
towards NH4
+-dominated
crop nutrition is possible?
Retention of soil-N in
agricultural soils is critical
for the sustainability of
production systems and to
prevent N from entering into
water-bodies
Nature 2013, 501:291
BNI function in plants should be exploited to facilitate retention of
soil-N within agricultural systems

56. We must develop new technologies to keep N to remain
and recycle within the agricultural systems and not
allow into water systems – Nitrification control is key
BNI function can be one such mechanism that can be
exploited from a breeding perspective and from a
system’s perspective
Take Home Message

57. Strategic Research Partner – CIAT
(Drs. IM Rao; Manabu Ishitani; John Miles; Joe Tohme; Jacobo Arango,
Marco Rondon; Maria Pilar Hurtado; Danillo Moreta; Gonzalo Borrero)
Other participating research institutes
ICRISAT (India)
CIMMYT (Mexico)
Tottori University (Japan)
Yokohama City University (Japan)
Scottish Crops Research Institute (UK)
Biogeochimie et ecologie des milieux continentaux (France)
CIAT
Tropical pastures-BNI
MAFF
GTZ
Forage-CRP(?)
JIRCAS
BNI Research
CIMMYT
Wheat-BNI
MAFF
Wheat-CRP
ICRISAT
Sorghum-BNI
MAFF (?)
Dryland cereals-CRP(?)
Thank you for the attention