Los días 20 y 21 de mayo de 2014, la Fundación Ramón Areces organizó el Simposio Internacional 'Microorganismos beneficiosos para la agricultura y la protección de la biosfera' dentro de su programa de Ciencias de la Vida y de la Materia.

1. Beneficial Microbes for Agriculture and Biosphere ProtectionCiencias de
la Vida y de la Materia | Madrid, 20 y 21 de mayo de 2014
F U N D A C I Ó N R A M Ó N A R EC E S
Comprometidos con el progreso, la investigación, la educación y la cultura
Session II. Microbe for sustainable agriculture: Biological Nitrogen Fixation
Biological Nitrogen Fixation
Tomás Ruiz Argüeso Centro de Biotecnología y Genómica de Planta
(CBGP) UPM-INIA. Madrid, Spain

2. Nitrogen is a limiting factor in Agriculture
Plants with and without N in a poor soil
Nitrogen plays a major role in the production of food and has conditioned the life of the
humanity since its origin.

3. Properties of N
• N2 comprises 78.3% of the earth's
atmosphere by volume (75.5% by mass).
• Two stable isotopes, 14N (99.6%) and 15N
(0.4%).
• N2 is chemically unreactive at the
temperatures and pressures of the
hydrosphere, biosphere, and atmosphere.
• N2 has a low solubility in water.
“Too Much or Too Little of a Good Thing”

4. NITROGEN FIXATION
• Non-biological process: electric storms,volcanic eruptions.
• Industrial N2 fixation: Haber-Bosch process 300-400 ºC, 500 atm, H2,
Fe catalyzer.
• Biological Nitrogen Fixation (BNF): Some prokaryotes are nitrogen-
fixing organisms
N N 2 NH3

5. Nitrogen and hydrogen are reacted over an iron catalyst
under conditions of 200 atmospheres, 450°C:
N2(g) + 3H2(g) 2NH3(g)
Fertilizer from Haber-Bosch process supports
40% of the world's human population
(Adapted from http://www.idsia.ch/~juergen/haberbosch.html)
*1898, Sir William Crookes, president of the British Association for the Advancement of
Science: “The world is running out of nitrogen”
*
Chemical nitrogen fixation: the Haber-Bosch process
F. Haber before of experimental design
that allowed him to generate amonia from
H2 and N2
Carl Bosch - BASF
Carl Bosch - BASF

6.  Increased crop yield. Without fertilizers, 40% of the current
agricultural production would be lost.
 However, the extensive use of commercial fertilizers
contaminates the groundwater and consumes fossil-fuel
resources (2% of the world energy). Fertilizers represent 50% of
the costs incurred in modern agriculture.
Chemical Fertilizers: Pros and Cons
Ammonium nitrate doesn’t look
dangerous.

7. Eutrophication

8. ¿Will the Haber-Bosch process have a more
efficient replacement?
(Fritz Haber in the ceremony of Nobel prize concession in
1920)
It may be that this solution is not the final one.
Nitrogen bacteria teach us that Nature, with its
sophisticated forms of the chemistry of living matter,
still understands and utilizes methods which we do
not as yet know how to imitate.
Use of Biological nitrogen fixation as an alternative to
increase crop productivity.

9. Nitrogen fixation (nif) gene cluster of Klebsiella pneumoniae. Genes encoding the nitrogenase
component proteins, nifHDK, are shown on the left. Genes whose products are involved in nitrogen
fixation are color coded according to their functions.
N2 + 8e- + 8H+ + 16 ATP 2NH3 + H2 + 16 ADP + 16Pi
FeMo-cofactor
Requirements
- ATP
- Reducing Power
- Low [O2]
The BNF process is catalyzed by the nitrogenase enzyme

10. Diazotrophs are scattered across microbial taxonomic groups, mostly in the
Bacteria but also in the Archaea
Nitrogen fixing systems
I) Non-symbiotic or free living:
Chemiotrophic bacteria (e.g. Azotobacter, Clostridium)
Phototrophic bacteria (e.g. Rhodobacter, Cyanobacteria)
ii) Associative symbiotic (e.g. Azospirillum, Azoarcus)
iii) Endosymbiotic diazotrophs (e.g. Frankia and
rhizobia).
Three groups of diazotrophs

11. a) The gram-positive filamentous actinomycete (Frankia)
Endosymbiotic diazotrophs
Non-leguminous actinorhizal
plants from 8 different families
such as the Betulaceae ( Alnus
sp.)
b) The gram-negative proteobacteria (rhizobia)
Legume plants (Fabaceae) such as
alfalfa (Medicago sativa), and all
common legumes (peas, beans,,
etc).
Induce N2 fixation in root nodule-like structures
rhizobia

12. :
Botany
- Angiosperms (Rosales Order)
- Highly diversified (20.000 species, 750 genera)
- Leaves: : alternes, compounds, stipulates
- Flowers: hermafrodites, fused sepals and five petals
- Fruit (pod : legume)
- Many species form root nitrogen fixing nodules in
symbiosis with soil bacteria known as rhizobia
The Rhizobium-legume symbiosis: Leguminosae (Fabaceae)
Percentage of species nodulated (Subfamilies)
Papilionoideae (Medicago, Pisum, Trifolium, Lotus, Lupinus, 98%
Mimosoideae (Mimosa, Acaceae….) 90%
Cesalpinoideae 40%

13. Estimation of the contribution of N2 fixation by legumes to human economy
(Herridge et al. 2008)
Legume cultivated surface
Pulse and oilseed 186 Mha)
Pasture and fodder 110 Mha
N2-fixed 33–46 Tg N annually
Nominal value US $50–70 billion
Relevant caracteristics of Fabaceae family
Fabaceae (“legumes”) is the third-largest family of angiosperms as regard the number of species what gives
an advantages as a target group of
global plant diversity assessments (GLDA):
includes economically important crop plants
Whole-genome sequences already available
Medicago truncatula Gaertn. (http://www.medicago .org/genome),
Lotus japonicus (Regel) K. Larsen (http://www .kazusa.or.jp/lotus),
Glycine max (L.) Merr. (http://www.phyto zome.net soybean, Schmutz & al., 2010)
Glycine soja Siebold & Zucc. (Kim & al., 2010)

14. Rhizobium-legume symbiosis
Two life styles: Rhizobia populate both soil and nodule niches.
Symbiotic rhizobia able to live within root nodules in the plant host.
Saprophytic : rhizobia growing ex planta able to survive in soil and the rhizosphere for years
Rhizobia are proteobacteria able to induce root nodules in legumes
(Fabaceae)
Burkholderia
Cupriavidus
Moulin et al. Nature 411 (2001)
16S rDNAAllorhizobium
Azorhizobium
Bradyrhizobium
Mesorhizobium
Rhizobium
Sinorhizobium
Methylobacterium
Rhizobium trifolii

15. Legume-rhizobia symbiotic interaction
Nodule factor Lipo-chitin oligosaccharides (LCOs) : fucose
residue alpha-1,3-linked to the GlcNAc residue
Synthesized by the coorditated action of more than 20
genes nod, where NodD is the central regulador.
Legumes have the ability to enter into mutually beneficial symbioses rhizobia followwing
signal transduction process reminiscent of ancient plant-mycorrhizal fungi
communication programs
Histochemica X-gal staining
highlights rhizobia in blue
Root surface

16. 16
16
Indeterminate nodules
The nitrogen-fixing nodule hosts symbiotic Rhizobium bacteroids
(Two types: Indetermitas and determinates
I
II
III
IV
symbiosomes
Central N-fixing zone
(leghemoglobin)
Determinate nodules
symbiosomes
bacteroid
Peribacteroidal
membrane
Nodule section
peas

17. Udvardi and Poole, ARPP 2013
Transport and metabolism in an infected nodule cell
NCR peptides

18. Hup+
N2
NH4
+
N2ase
H2
ROOT
ENERGY
organic
N
HUP
H2
NH4 N2
+
ROOT
ENERGY
organic
N
N2ase
Hup--
Energetic efficiency of Rhizobium strains
N2+8H++8e-+16-24 ATP 2NH3+16-24 ADP+16Pi+ H2
H2
H+ + e-
Hydrogenase
Hydrogen Metabolism in Nodules

19. Hydrogenase of D. gigas
(Volbeda et al., 1995)
H2 --> 2H+ + 2 e-
HupL
HupS
Structure and function of the hydrogenase FeNi cofactor
of R. leguminosarum
2
Buried Ni-Fe
Active Site
hup
GS HF J KC B DD A EL F CI X
hyp
E
- 18 genes required for for synthesis of NiFe hydrogenase
- 10 genes are induced for NiFe cofactor synthesis and assembly
H
2
NiFe cofactor
Hydrogenase gene cluster

20. Symbiotic nitrogen fixation
Hydrogen oxidation
hup genes hyp genes
FnrNNifA
O2
control
fixNOQP
fixGHIS
nif and fix
genes
FixLJK
other
metabolic
activities

21. Coexpression of nitrogenase and hydrogenase in pea nodules

22. Hydrogenase increases symbiotic efficiency of rhizobia strains
2.- Generation of Hup+ strains (TnHup):
Vicia sativa/R. leguminosarum symbiosis (J. Sanjuan and our group)
1.-Comparison of Hup+/Hup- strains:

23. Role of BNF in survival of endangered legume species
Fabaceae display a range of rarity (abundance of individuals or range size) ,
from extreme endemics (only known from small local areas, which are
exceedingly vulnerable to threats),
to widespread and even cosmopolitan species
Understanding the natural history of rare plants is crucial to their conservation.
It has long been recognized that basic biological knowledge of a species can help
to identify factors that limit long-term persistence.

24. An endangered legume plant: Lupinus mariae-josephi (Pascual 2004)

25. Native lupine species from the Iberia Peninsula
L. angustifolius
L. cosentinii
L. hispanicus
L. luteus
L. micranthus
L. gredensis
L. albus
L. polyphyllus
Native Lupinus spp. : L. angustifolius, L. cosentinii, L. hispanicus, L. gredensis, L. luteus, L. micranthus.
Naturalized or introduced lupines: L. albus, L. polyphyllus

26. Phylogeny of Lupinus spp. (L. mariae-josephae)
New data and phylogenetic placement of the enigmatic Old World lupin: Lupinus mariae-josephi H. Pascual.
Mahe et al. Genet Resour Crop Evol (2011) 58:101–114

27. Endemism of L. mariae-josephae

28. Samples from
four different
areas in
Valencia
(Spain)
L. mariae-josephae
trap plants
1 -10 nodules per plant
YMA plate
PCR-RAPDs 19 different profiles Lmj strains
M m n l a i g b d k s r e o q p h j c f pac M
Isolation of L. mariae-josephae endosymbiotic bacteria
Location
Number
of isolates
(N)
Number
of RETs
(S)
RETs
(number of
isolates)
Richness
(S/N)
Diversity
(h)
Soil Chemical
Characteristics
pH CaCO3 (%)
Llombai
34 8
a(2), b(5), c(5),
d(5), e(4), f(4),
g(4), h(5)
0,24 0,89 7.98 12
Monserrat 16 5
k(2), l(4), m(5),
n(2), o(3)
0,31 0,82 8.14 14
Xativa
27 2 p(19), q(8) 0,07 0,43 8.17 18
Gandia
26 4
r(6), s(11), i(4),
j(5)
0,15 0,73 7.88 9
Sánchez-Cañizares et al. (2011) Syt. Appl. Microbiol. 34, 207-2011
Durán et al. (2013) Syst. Appl. Microbiol. 36, 128– 136

29. Legume hosts Nodulacion y fijación de N2
LmjA2 LmjB2b LmjC LmjM3 LmjM6
Lupinus mariae-josephae Yes + high Yes + high Yes + high Yes + high Yes + high
L. angustifolius No No No No No
L. luteus No No No No No
L. hispanicus No No No No No
L. gredensis No No No No No
Ornithopus compressus No No No ND ND
L. micranthus Yes + poor No Yes + poor Yes + high Yes + high
L. cosentinii Yes + poor Yes + poor Yes + high Yes + No Yes + No
L. albus Yes + poor Yes + poor Yes + high ND ND
Vigna sinensis Yes + No Yes + No Yes + No ND ND
Macroptilium atropurpureum No No Yes + poor Yes + high Yes + high
L. angustifolius / Lmj strainsVigna sinensis / Lmj strains L. mariae-jospeshae / Lmj strains LmjC nodules
Cross-inoculation assays

30. LmjG2
LmjG3
LmjD32
LmjL9
LmjH2p
LmjX7
LmjL7
LmjM10
LmjTA6
LmjX10
LmjB2b
LmjTA10
LmjM3
LmjM1
LmjM2
LmjM6
LmjA2
B. lablabi CCBAU 23086T
B. pachyrhizi PAC48T
B. jicamae PAC68T
B. elkanii USDA76T
LmjC
LmjL5
clade I
B. liaoningense LMG18230T
B. yuanmingense CCBAU 10071T
B. daqingense CCBAU 15774T
B. canariense BTA-1T
B. japonicum USDA6T
B. denitrificans LMG 8443T
B. huanghuaihaiense CCBAU 23303T
B. iriomotense EK05T
B. betae PL7HG1T
B. rifense CTAW71T
B. cytisi CTAW11T
clade II
Bosea thiooxidans DSM9653
99
87
100
95
100
82
0.01
16S rDNA
Neighbor Joining phylogenetic tree showing
relationships of nodule isolates from L. mariae-
josephae and other symbiotic bacterial strains
based on nearly complete 16S rDNA gene
sequences (1,400 bp). Bootstrap values (greater
than 70%) were calculated for 1,000 subsets and
are indicated at relevant nodes.
Bradyrhizobium genus

31. Neighbor Joining phylogenetic tree based on
the alignment of a concatenated nucleotide
sequence of glnII, recA and atpD. NJ
bootstrap support values (≥ 70% over 1,000
replicates) are indicated at relevant nodes
Lmj M.3
Lmj M.6
Lmj M.2
Lmj A2
Lmj Ta.6
Lmj L.7
Lmj B2b
Lmj D32
Lmj M.1
Lmj G.2
Lmj G.3
Lmj H2p
Lmj M.10
Lmj X.7
Lmj X.10
Lmj L.9
Lmj Ta.10
Lmj C
Lmj L.5
B. jicamae strain PAC68
B. lablabi CCBAU 23086
B. elkanii USDA 76
B. pachyrhizi PAC48
B. betae LMG 21987
B. japonicum USDA 6
B. canariense LMG 22265
B. cytisi CTAW11
B. iriomotense LMG24129
B. liaoningense LMG 18230
B. yuanmingense LMG 21827
S. meliloti 1021
100
100
85
53
84
98
99
100
92
100
99
100
96
85
70
71
88
94
99
99
68
78
89
52
75
98
0.000.020.040.060.080.10
A1
C
B
E
D
F
95 %
OTUs
(Operational Taxonomic Units)
glnII, recA, atpD
A2
This phylogenetic tree reveals a high
degree of heterogenity, higher within
Bradyrhizobium genus

32. Nodulation genes: Phylogenetic analysis shows a high degree of homogenity
LmjA2
LmjX7
LmjH2p
LmjX10
LmjC
LmjL5
LmjL9
LmjB2b
LmjD32
LmjL7
cluster A
LmjM3
LmjM6
LmjM2
LmjM1
LmjG2
LmjTA10
LmjG3
LmjM10
LmjTA6
B. lablabi CCBAU 23086 T
cluster B
B. jicamae PAC68T
B. iriomotense EK05 T
R. leguminosarum bv. viciae 3841
S.meliloti 1021
B. liaoningense LMG 18230 T
B.canariense BTA-1T
B. cytisi CTAW11T
B. elkanii USDA 76T
B. pachyrhizi PAC48T
B. yuanmingense CCBAU 10071T
B. japonicum USDA6 T
B. huanghuaihaiense CCBAU 23303 T
B. daqingense CCBAU 15774 T
A. caulinodans ORS 571
100
100
97
92
83
100
100
99
87
99
97
86
84
79
78
0.1
LmjX7
LmjX10
LmjH2p
LmjC
LmjA2
LmjB2b
LmjD32
LmjL7
LmjL5
LmjL9
cluster A
LmjTA10
LmjTA6
LmjG3
LmjG2
LmjM10
LmjM1
LmjM2
LmjM6
LmjM3
B. lablabi CCBAU23086T
cluster B
B. jicamae PAC68T
B. iriomotense EK05T
B. elkanii USDA76T
B. yuanmingense CCBAU 10071T
B. japonicum USDA6 T
S. meliloti 1021
R. leguminozarum bv. viciae 3841
A. caulinodans ORS571
99
100
100
96
71
100
81
99
100
88
80
51
100
80
71
100
100
0.05
nodC nodA
Neighbor Joining phylogenetic tree based on partial nodC and nodA sequences. The scale bar shows the number of
substitutions per site. The same grouping of strains were observed with 12 other nod genes

33. B. valentinum LmjM3 sp. nov. (L. mariae-josephae)
Bradyrhizobium valentinum sp. nov., isolated from effective
nodules of Lupinus mariae-josephae, a lupine endemic of basic-
lime soils in Eastern Spain
Durán et al. (2014)
WT LmjM3
Control

34. The Rhizobium symbiosis is a key factor for conservation
of the endangered
Lupinus mariae-josephae species

35. Reproducing plants for conservation purposes
The Lmj-nodulating bradyrhizobia are at low densities in the “terra rossa” soils of
Valencia region.
This paucity may contribute to the lack of success in reproducing plants for
conservation purposes.
Two strains, LmjC and LmjM3, were selected as inocula for seed coating.
Two planting experiments were carried out in consecutive
years(2012 and 2013)

36. Conservation efforts: L. mariae-josephae field experiments
Peat coated pre-germinated seeds
Ripe plant containing pods
Area of field experiment (near Llombai patch)
Emerging cotyledons

37. Effect of inoculation in plant survival, and pod and grain yields of L. mariae-
josephae in field trials performed in two successive seasons
Albert et al. 2014

38. Endangered legume species
In the latest version of the Red List of IUCN (IUCN, 2012) (International Union for Conservation of
Nature and Nature Resources) , 837 species of Fabaceae were assessed and 75% are assigned to the
categories: Extinct (EX, 6 spp.), Extinct in the Wild (EW, 1 sp.), Critically Endangered (CR, 74 spp.),
Endangered (EN, 165 spp.), and Vulnerable (VU, 378 spp.). Only 4% “(837/19400)” of all legume
species have been assessed.
GLDA would contribute to fill this gap by organizing a project for assessing most legume species in the
world under IUCN criteria.
IUCN
((International Union for Conservation of Nature and Nature Resources)
Most currently IUCN critera to define rare small range legume species are based on history of
reproductive traits such as number of pods, seeds
The examination of the N2 fixing Rhizobium-legume symbiosis has never been counted as a
factor for legume conservation.
Our results based on Lmj seed inoculation indicate that
the presence of N2-fixing efficient Rhizobium strains is a
relevant factor to consider in the conservation of other
endanger legume species

39. UPM-CBGP, Madrid
David Durán Wendt
Carmen Sánchez Cañizares
Luis Rey
José Manuel Palacios
Juan Imperial
Tomás Ruiz-Argüeso
Francisco Temprano
IFAPA Las Torres-Tomejil. Alcalá del Rio (Sevilla)
Esperanza Martínez-Romero
Centro de Investigación sobre Fijación de Nitrógeno, UNAM.
Ap Postal 565 – A. Cuernavaca, Morelos MÉXICO, D.F.
Abdelkader Ainouche
UMR CNRS 6553 Ecobio, Université de Rennes-1 Rennes, France
Albert Navarro
Centro de Investigación y Experimentación Forestal Quart de
Poblet (Valencia)
Supported by
Fundación BBVA
Comunidad de Madrid
MICINN 2011
AECID 2012
Universidad Politécnica
de Madrid-CBGP
Madrid (Spain)
CENTRO DE BIOTECNOLOGÍA Y
GENÓMICA DE PLANTAS
ETSIA
Dinitrogen fixing endosymbiosis of Lupinus mariae-josephae, a unique lupine species
endemic of basic soils of Eastern Spain