Biological Nitrification Inhibition (BNI) in Plants – Implications for Improving Nitrogen-use efficiency and

Reducing Nitrous oxide Emissions from Agricultural SystemsA Genetic-Mitigation Strategy

GV SubbaraoJIRCAS, JapanGV SubbaraoJIRCAS, Japan

CollaboratorsCIAT

CIMMYTICRISAT

Tottori University

Participation from JIRCAST. Ando

K. NakaharaT. YoshihashiT. Ishikawa

BNI-Mitigation Technology

Team

Year1950 1960 1970 1980 1990 2000 2010 2020

Nitr

ogen

eff

icie

ncy

in c

erea

l pro

duct

ion

(meg

a to

nnes

cer

eal g

rain

/meg

aton

ns fe

rtiliz

er a

pplie

d)

20

30

40

50

60

70

80

Why NUE is <30% in most agricultural systems?

Uncontrolled rapid nitrification in agricultural soils

NUE (Nitrogen use efficiency)

Global food production has doubled since the advent of Green Revolution

(1960 – 2000), but Nitrogen fertilizer consumption has increased 10-fold

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 applications have reached a point of

diminishing returns i.e. we are applying more andmore nitrogen to get similar yieldsand this may continue in future

N-fertilization efficiency is declining in cereal production

Nitrogen fertilizer consumption worldwide in 2010

>120 Tg (million metric tons)

Energy cost of nitrogen fertilizer – 1.8 to 2 L diesel oil per kg N fertilizer

1.70 billion barrels of diesel oil (energy equivalent) is needed to produce this nitrogen

fertilizer

BNI Concept

Ammonium(NH4+)

Nitrite(NO2-)

Nitrate(NO3-)Ammonia-oxidizing Bacteria Nitrite-oxidizing BacteriaBL

BL

BL BL

Microbial-N

Imm

obili

zatio

n

Min

eral

izat

ion

Nitrate leaching

Denitrification

N2O, NO and N2

N lost from agricultural system

N lost from agricultural system

Concept of Biological Nitrification Inhibition (BNI)

BNI-Mitigation Technology?FAO workshop on "The Role of Agricultural

Biotechnologies in Sustainable Food Systems and Nutrition", 15-17 Feb. 2016,

Rome, Italy

Plants release two categories of BNIs

Hydrophobic Hydrophilic

BNI Activity

Mostly confined toRhizosphere

May move out of Rhizosphere

Plant -rootproduced

nitrificationinhibitors

BL

BL

BL

BL

BL

BL

BL

BL

BL

BL

BL

BL

BL

BLBL

BL

BL

BL

BL BNI-Mitigation Technology

Subbarao G V et al. PNAS 2009;106:17302-17307

©2009 by National Academy of Sciences

Brachiaria pastures release a powerful nitrification inhibitor from root systems

Chemical structure of

BrachialactoneA tricyclic terpenoid with a unique 5-8-5

membered ring system and a g-lactone ring

B. humidicola

FAO workshop on "The Role of Agricultural Biotechnologies in Sustainable Food

Systems and Nutrition", 15-17 Feb. 2016, Rome, Italy

BNI-Mitigation Technology

Brachialactone’s mode of inhibitory action on Nitrosomonas

CompoundConcentration in the in vitro assay, mM AMO pathway HAO pathway

Crude-root exudate (methanol extract) 63.4 + 0.8 63.8 + 0.8Brachialactone 5.0 59.7 + 0.9 37.7 + 0.9Nitrapyrin 3.0 82.3 + 1.5 8.1 + 1.2

Inhibition (%)

Outer Membrane

Inner Membrane

Periplasm

Nitrosomonas

mM

Brachialactone

Brachialactone–HAO Docking simulation

Receptor： N.oceani HAOLigand： Brachialactone

Autodock vina 1.1.2

Heme P460

Brachialactone

Docking simulation hypothesized that brachialactone could bind to the gateway of the ligand pocket.

Brachialactone is too large to bind to the reaction center.

Nishigaya, NIASJIRCAS-NIAS collaboration

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 mg 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

Does it work in the field?FAO workshop on "The Role of Agricultural

Biotechnologies in Sustainable Food Systems and Nutrition", 15-17 Feb. 2016,

Rome, Italy

BNI-Mitigation Technology

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 fieldCan BNI function in plants be exploited to develop low-N2O emitting systems then?

BNI capacity in root systems – Field plots

No BNIcapacity

Highest BNIcapacity

BNI-Mitigation Technology

Photo: J. W. Miles

Characterization of residual effect of BNI from B. humidicola pasture on maize productivity and Nitrogen use efficiency (2012-2015)

Suppressing soil nitrification with BNI-enabled Brachiaria pastures

JIRCAS-CIAT partnership

Can it improve NUE in maize when it is planted after Brachiaria pasture?

BNI-Mitigation Technology

120 kg N ha-1

BNI-Field Non-BNI-Field

120 kg N ha-1

Nitrogen Fertilizer Applied (kg N ha-1)

0 50 100 150 200 250 300

Mai

ze G

rain

Yie

ld (k

g ha

-1)

0

1000

2000

3000

4000

5000

6000

BH-BNICropped-No-BNI

BNI-Field

Non-BNI-FieldHigh-nitrifying field

4th year of maize crop after removing BH

Nitrogen Fertilizer Applied (Kg N ha-1)

40 60 80 100 120 140 160 180 200 220 240 260

Agr

onom

ic N

itrog

en U

se E

ffic

ienc

y(K

g G

rain

yie

ld K

g-1 N

app

lied)

10

15

20

25

30

35

40

45

BH-BNICropped - No-BNIBNI-Field

4th year of maize crop after removing BH

Non-BNI-FieldHigh-nitrifying field

Maize yields and NUE are substantially higher in BNI-fields compared to non-BNI

fields

A healthy maize crop N-deficient maize crop

Proof of concept for BNI function in sorghum A field study at ICRISAT

JIRCAS-ICRISAT partnership

FAO workshop on "The Role of Agricultural Biotechnologies in Sustainable Food

Systems and Nutrition", 15-17 Feb. 2016, Rome, Italy

O

O

OH

O 63-66 Mbp

Sorghum roots release a nitrification inhibitor from roots

Sorgoleone

Sorgoleone production is controlled by chromosome 1

JIRCAS-ICRISAT partnership

Chromosome-1

A Genetic-Mitigation Technology?

200N 200N+Sorgoleone 200N+DCD0

0.20.40.60.8

11.21.41.61.8

Integrated N2O emission

Treatments

N2O

em

issio

n (u

g N

2O-N

)

a

b

c

2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44-8-327

121722273237

N2O emission

200N200N+Sorgoleone200N+DCD

Days of incubationN2O

em

issio

n (n

g N

2O-N

(g d

ry so

il)-1

hr-

1 m

-2)

Sorgoleone additions to the soil suppressed N2O emissions

BNI-Mitigation Technology

Genetic improvements in sorgoleone release from sorghum root systems lead to lower N2O emissions?

Introducing high-BNI capacity into wheat Developing low-nitrifying and low-N2O emitting wheat production systems

JIRCAS-CIMMYT partnership

FAO workshop on "The Role of Agricultural Biotechnologies in Sustainable Food

Systems and Nutrition", 15-17 Feb. 2016, Rome, Italy

Plant species0 1 2 3 4

BN

I act

ivity

rele

ased

from

root

s(A

TU g

-1 ro

ot d

ry w

t. d-1

)

0

5

10

15

20

25

30

35

NH4-N grownNO3-N grown

Nobeoka Chinese Spring

L. racemosus

Wild-wheat has high-BNI capacity

Leymus racemosus

Leymus does not release BNI when grown with NO3

--N where pH of RE-collection solution will be of >6.0

RE collection

pH 4.0

RE collection

pH 7.5

JIRCAS-CIMMYT partnership

A Genetic-Mitigation Technology?

FAO workshop on "The Role of Agricultural Biotechnologies in Sustainable Food

Systems and Nutrition", 15-17 Feb. 2016, Rome, Italy

n

N-inputs into Agriculture

175 Tg N(120 Tg N from N-

Fertilizers + N-fixed from legumes)

Plant protein-N53.5 Tg

Nitrogen flow in Human-centric Ecosystems (where most ecological components are

directed to serve human society)

AnimalProtein-N3.5 Tg Human-N

0.3 Tg

123.5 Tg N

LostAgriculture

48.0 Tg N Lost

Livestock5.0 Tg N LostSewage systems

N2O, NO, N

2

NO3

- leaching

If there were no N losses from agricultural systems and recycle most of the N excreted from animals (i.e.

manure) only a small fraction of N needs to be replaced by fertilizer N inputs annually

FAO workshop on "The Role of Agricultural Biotechnologies in Sustainable Food

Systems and Nutrition", 15-17 Feb. 2016, Rome, Italy

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 The negative consequences of Green Revolution?

FAO workshop on "The Role of Agricultural Biotechnologies in Sustainable Food

Systems and Nutrition", 15-17 Feb. 2016, Rome, Italy

Year1850 1900 1950 2000 2050 2100 2150

N2O

Em

issi

ons i

n Tg

0

2

4

6

8

10

12

14

16

18

AnthropogenicNatural

Projected

Nitrification and denitrification are the primary drivers for N2O emissions from agricultural systems and account for >70% of global N2O emissions

Green Revolution

IPCC set a target to cut GHG emissions by 2050 to limit global temperature raise to

<2CA requirement set by COP-21

–The Paris Agreement

There is an urgency to develop next-generation technologies to reduce N2O emissions from agricultural systems

Exploiting BNI-Mitigation Technology could be one such powerful strategy

FAO workshop on "The Role of Agricultural Biotechnologies in Sustainable Food

Systems and Nutrition", 15-17 Feb. 2016, Rome, Italy

Based on Khalil and Rasmussen 1988Annals of Glaceology 10:73-79

Year1880 1900 1920 1940 1960 1980 2000 2020 2040 2060

N2O

con

cent

ratio

n in

the

atm

osph

ere

(ppb

v)

280

300

320

340

360

380

400

N2O concentration in atmosphere

(ppbv)

The Choices We Make Will Create Different Outcomes

With substantial mitigation

Without additional mitigation

Change in average surface temperature (1986–2005 to 2081–2100)

Source: IPCC AR5 synthesis report

FAO workshop on "The Role of Agricultural Biotechnologies in Sustainable Food

Systems and Nutrition", 15-17 Feb. 2016, Rome, Italy

FAO workshop on "The Role of Agricultural Biotechnologies in Sustainable Food

Systems and Nutrition", 15-17 Feb. 2016, Rome, Italy

Benefits from Genetic-Mitigation using BNI-Technology

It will not require expensive or cumbersome management changesNo additional cost to the farmers once BNI trait is introduced into staple crops/pasturesLarge-scale impacts are potentially achievable as adoption is only through seedDelivery of BNIs through root systems is more effective than synthetic nitrification inhibitors as fertilizer additivesNo negative environmental consequencesNo health issues on food or feed qualityCan bring long-term benefits by changing potential soil nitrification, microbial-ecologyIf properly integrated into cropping systems, BNI-technology can improve soil-N retention and contribute to the sustainability of production systems

1st International BNI Workshop held during 2-3rd March 2015 at JIRCAS, Tsukuba, Japan

FAO workshop on "The Role of Agricultural Biotechnologies in Sustainable Food

Systems and Nutrition", 15-17 Feb. 2016, Rome, Italy