Maximizing the N2 fixation and minimizing the N20 emissions in alfalfa production using natural inoculants

Dr. Nicolás Ayub

National Scientific and Technical Research Council - Argentina National Institute of Agricultural Technology - Argentina

Nitrogen-fixing alfalfa inoculants: low cost biofertilizers which reduce our dependence of the petroleum

inoculant-treated alfalfa plants grow without

nitrogen fertilizers derived from non-renewable fossil

fuels, and add fixed nitrogen to the soil

INOCULANT

10% COAT

10% COAT APRON SEED TREATMENT ALFALFA SEED

N2

NH3

N-org

NO3-

N-org

N-org

active nodule

senescent nodule

coated seeds

Haber-Bosch

NH3

synthetic fertilizers

H2 + N2

metal catalyst (500°C, 300 atm)

non-legume crops

INOCULANT

Commercial alfalfa inoculants were domesticated prior to the emergence of global environmental change

N2 → NH3 NO3- → N2O

potential activities of alfalfa rhizobia

++++ proteins

high-N soil

low-N soil

high N2O emissions

lights shadows? and

N-fixing simbiosis

incomplete denitrification

domestication of efficient nitrogen-fixing strains for alfalfa (1960/70s)

the work in the area of climate change began in the late 1980s

model strains (e.g.

1021) produce (NAP+, NIR+ and NOR+) and

degradate (NOS+) greenhouse gas N2O NO3

-

NO2-

NO

N2O

NAP+

NIR+

N2

NOR+

NOS+

commercial alfalfa inoculants (e.g. B399=102F34 and B401) ?

Is there a reasonable balance between economic and environmental performance ?

Genomic analyses of non-commercial (NC: GR4, Rm41 and 1021) commercial (C: B399 and B401) strains

commercial strains suffered a 15-kb deletion in the

megaplasmid A region that contain the NOS cluster

(nosRZDFYLX)

gen

om

e s

ize

(p

b)

C NC NC C NC C NC C

OR

F (#

)

fram

esh

ifte

d

gen

es

(#)

pse

ud

oge

ne

s (

#)

NC

nosRZDFYLX

1021

Rm41

GR4

B399 B401

15-kb deletion

C

1021

B399

B401

napCBAD nirK norDQBCE

C

NC

G A

SMa1178 SMa1200 nosD

BWO90_02885 (pseudogene)

BWO90_02890 (frameshifted gene)

deletion 1 deletion 2

1021

B399

GCGAACGACACGCCGGGCGCTCGGCTGAACGACACGCCGGGAGCCGGAAACTCGAGATCGTCGCCGCTCTAACCTTCGGCC

nonsense mutation

premature stop codon

650399-650421 (nosD)

B399

660549-660596 (SMa1200)

645443-645452 (SMa1178)

traces of nos genes

in B399

Environmental risk of the rhizobial domestication

N2

N-org

N-org

N2

N2O

NOS-

NOS+

N2O N2O

NO3-

NO2-

NO

NAP+

NIR+

NOR+

senescent

nodule active nodule

N2O

N2 N2 N2 N2 N2 N2 N2 N2 N2 N2 N2 N2 N2

NH3

N-org

rhizobial domestication

our proposal (back to the soil)

non-domesticated rhizobes (NC strains) domesticated rhizobes (C strains)

rhizobial domestication

New rizobial strains (NRS) derived from alfalfa nodules containing both N-fixation and N2O reductase activities

B399 (positive control)

NRS7 NRS15 uninoculated (negative control)

Estimation of N-fixation ability by hydroponic cultures (100% vermiculite + minimal medium INTA13)

quantification of N2O emissions by microcosm system

(soil inoculation + gas chromatography)

PCR colony assays aqpZ+ → + Ensifer meliloti nosZ+ → + N2O reductase

Identification of several NAP and NIR mutants

Selected strains for maximizing economic and environmental benefits:

NO3- NO2

- NO N2O N2 NAP+ NIR+ NOR+ NOS+ NO2

- NO N2O N2 NAP- NIR+ NOR+ NOS+ NO N2O N2 NAP- NIR- NOR+ NOS+

NO3- NO2

- NO N2O N2 NAP+ NIR- NOR+ NOS+

1) 2) 3)

4)

↑ N content (NO3- and NO2

-) in the soil (due to NAR-/NIR-)

↑ degradation of N2O in the soil (due to NOS+)

↓ production of N2O in the soil (due to NAR-/NIR-)

Thanks

nicoayub@gmail.com

ayub.nicolas@inta.gob.ar

https://www.researchgate.net/profile/Nicolas_Ayub