Co-inoculation of a Pea Core-Collection with Diverse Rhizobial Strains Shows Competitiveness for Nodulation and Efficiency of Nitrogen Fixation Are Distinct traits in the Interaction

Abstract

Pea forms symbiotic nodules with Rhizobium leguminosarum sv. viciae (Rlv). In the field, pea roots can be exposed to multiple compatible Rlv strains. Little is known about the mechanisms underlying the competitiveness for nodulation of Rlv strains and the ability of pea to choose between diverse compatible Rlv strains. The variability of pea-Rlv partner choice was investigated by co-inoculation with a mixture of five diverse Rlv strains of a 104-pea collection representative of the variability encountered in the genus Pisum. The nitrogen fixation efficiency conferred by each strain was determined in additional mono-inoculation experiments on a subset of 18 pea lines displaying contrasted Rlv choice. Differences in Rlv choice were observed within the pea collection according to their genetic or geographical diversities. The competitiveness for nodulation of a given pea-Rlv association evaluated in the multi-inoculated experiment was poorly correlated with its nitrogen fixation efficiency determined in mono-inoculation. Both plant and bacterial genetic determinants contribute to pea-Rlv partner choice. No evidence was found for co-selection of competitiveness for nodulation and nitrogen fixation efficiency. Plant and inoculant for an improved symbiotic association in the field must be selected not only on nitrogen fixation efficiency but also for competitiveness for nodulation.

Full text

ORIGINAL RESEARCH
published: 10 January 2018
doi: 10.3389/fpls.2017.02249
Frontiers in Plant Science | www.frontiersin.org 1January 2018 | Volume 8 | Article 2249
Edited by:
Brigitte Mauch-Mani,
University of Neuchâtel, Switzerland
Reviewed by:
Sharon R. Long,
Stanford University, United States
Ton Bisseling,
Wageningen University & Research,
Netherlands
*Correspondence:
Virginie Bourion
virginie.bourion@inra.fr
Marc Lepetit
marc.lepetit@inra.fr
In Memoriam:
This contribution is dedicated to the
memory of Dr. Gisèle Laguerre, who
initiated this work and through her
activities raised interest in rhizobial
resources as the foundation for
improved legume agriculture.
Specialty section:
This article was submitted to
Plant Microbe Interactions,
a section of the journal
Frontiers in Plant Science
Received: 29 June 2017
Accepted: 21 December 2017
Published: 10 January 2018
Citation:
Bourion V, Heulin-Gotty K, Aubert V,
Tisseyre P, Chabert-Martinello M,
Pervent M, Delaitre C, Vile D, Siol M,
Duc G, Brunel B, Burstin J and
Lepetit M (2018) Co-inoculation of a
Pea Core-Collection with Diverse
Rhizobial Strains Shows
Competitiveness for Nodulation and
Efficiency of Nitrogen Fixation Are
Distinct traits in the Interaction.
Front. Plant Sci. 8:2249.
doi: 10.3389/fpls.2017.02249
Co-inoculation of a Pea
Core-Collection with Diverse
Rhizobial Strains Shows
Competitiveness for Nodulation and
Efficiency of Nitrogen Fixation Are
Distinct traits in the Interaction
Virginie Bourion 1
*, Karine Heulin-Gotty 2, Véronique Aubert 1, Pierre Tisseyre2,
Marianne Chabert-Martinello 1, Marjorie Pervent 2, Catherine Delaitre 1, Denis Vile 3,
Mathieu Siol 1, Gérard Duc 1, Brigitte Brunel 2, Judith Burstin 1and Marc Lepetit 2
*
1Agroécologie, INRA, AgroSup Dijon, Université Bourgogne Franche-Comté, Dijon, France, 2Laboratoire des Symbioses
Tropicales et Méditerranéennes, INRA, IRD, CIRAD, Montpellier SupAgro, Université de Montpellier, Montpellier, France,
3Laboratoire d’Ecophysiologie des Plantes Sous Stress Environnementaux, INRA, Montpellier SupAgro, Université de
Montpellier, Montpellier, France
Pea forms symbiotic nodules with Rhizobium leguminosarum sv. viciae (Rlv). In the field,
pea roots can be exposed to multiple compatible Rlv strains. Little is known about the
mechanisms underlying the competitiveness for nodulation of Rlv strains and the ability of
pea to choose between diverse compatible Rlv strains. The variability of pea-Rlv partner
choice was investigated by co-inoculation with a mixture of five diverse Rlv strains of
a 104-pea collection representative of the variability encountered in the genus Pisum.
The nitrogen fixation efficiency conferred by each strain was determined in additional
mono-inoculation experiments on a subset of 18 pea lines displaying contrasted Rlv
choice. Differences in Rlv choice were observed within the pea collection according to
their genetic or geographical diversities. The competitiveness for nodulation of a given
pea-Rlv association evaluated in the multi-inoculated experiment was poorly correlated
with its nitrogen fixation efficiency determined in mono-inoculation. Both plant and
bacterial genetic determinants contribute to pea-Rlv partner choice. No evidence was
found for co-selection of competitiveness for nodulation and nitrogen fixation efficiency.
Plant and inoculant for an improved symbiotic association in the field must be selected
not only on nitrogen fixation efficiency but also for competitiveness for nodulation.
Keywords: partner choice, competitiveness for nodulation, pea-rhizobium symbiosis, nitrogen fixation efficiency,
genetic diversity, plant breeding, Pisum sativum,Rhizobium leguminosarum sv. viciae
INTRODUCTION
Legumes are a sustainable source of protein for both human and animal diets. Owing to their
ability to establish symbiosis with nitrogen-fixing bacteria, their cultivation is vital for reducing
the use of nitrogen fertilizers, a major cause of agricultural greenhouse gas emissions and energy
consumption (Jensen and Hauggaard-Nielsen, 2003; Galloway et al., 2008). The biological nitrogen

Bourion et al. Genetic Diversity of Pea-Rhizobium leguminosarum Partner Choice
fixation (BNF) obtained from grain legume crops (pulses and
oilseed legumes) represents a quarter of the N applied to
arable lands annually as chemical fertilizers (Herridge et al.,
2008). Despite these benefits, grain legumes are under-cultivated
in European agricultural systems. They suffer from lower
productivity and more variable yields than cereals whose
production depends on high fertilizer inputs. Improving the
regularity of grain legume yield is thus a major objective from
an agroecological standpoint, and one avenue to achieve it could
be through improved symbiosis.
Pea (Pisum sativum L.) is one of the world’s most cultivated
pulse crops (Duc et al., 2015). It was one of the first domesticated
crops and was an important component, along with other
legumes and cereals, of the diet of early civilizations in the Middle
East and the Mediterranean Basin (Zohary and Hopf, 1973;
Cousin, 1997; Smykal et al., 2015). Wild pea relatives include the
species P. fulvum and P. sativum subsp. elatius. Cultivated pea
appeared in the early Neolithic period in the area of the Fertile
Crescent, and later spread throughout Europe, through Turkey,
Greece and the Caucasus, and eastwards to India and China
through Iran and Afghanistan. Cultivated peas mostly belong to
P. sativum subsp. sativum.P. sativum subsp. abyssinicum is a
less frequently cultivated pea, restricted to Yemen and Ethiopia
(Vershinin et al., 2003; Jing et al., 2010; Smykal et al., 2011).
Establishment of the symbiosis involves mutual recognition
of the plant legume and rhizobial partners in the soil, followed
by the development of root symbiotic organs called nodules
in which the bacteria fix dinitrogen. In most cases, the
symbiotic interaction between pea and rhizobia involves strains
of Rhizobium leguminosarum symbiovar viciae (Rlv). Rlv has long
been regarded as able to nodulate all the species of the legume
tribe Viciae. However, differences in symbiotic host range within
the Viciae tribe have been reported between Rlv strains (Laguerre
et al., 2003; Mutch and Young, 2004). Sequence variation in the
Rlv symbiotic nod genes involved in Nod factor production has
been described (Laguerre et al., 2001; Kumar et al., 2015; Peix
et al., 2015). Variation of Rlv host specificity within the genus
Pisum was one of the earliest reported cases of host-controlled
restriction of nodulation. Some cultivated peas from Afghanistan
and Middle East were identified as being resistant to nodulation
by European Rlv strains, and requiring specific Rlv strains
found in Israel, Turkey or Afghanistan to nodulate, whereas
European pea cultivars were nodulated by both European and
Middle Eastern strains when mono-inoculated (Lie, 1978; Young
and Matthews, 1982). The pea SYM2 and bacterial nodX genes
confer this specific interaction (Holl, 1975; Davis et al., 1988).
Such variations in both bacterial nod genes and legume genes
encoding receptor-like kinases (LysM-RLKs) required for Nod
factor perception indicate them to be major determinants of the
specificity of a legume-rhizobia association (Spaink et al., 1991;
Denarie et al., 1992; Walker et al., 2000; D’Haeze and Holsters,
2002; Liang et al., 2014).
In the complex soil environment, legume roots are exposed
to heterogeneous rhizobial populations containing multiple
compatible strains (Laguerre et al., 2003; Mutch and Young,
2004; Bourion et al., 2007; Sachs et al., 2009). Various data
indicate that BNF could be suboptimal in pea as natural Rlv
populations are quantitatively and qualitatively heterogeneous,
which often results in nodulation of peas by poorly efficient
rhizobia (Fesenko et al., 1995; Laguerre et al., 2007). There is
general agreement about the interest of rhizobial inoculation of
pea with selected Rlv bacteria for improving BNF (Bremer et al.,
1988; Fesenko et al., 1995; McKenzie et al., 2001). However, even
when pea seeds are inoculated with efficient Rlv strains, these
may be outcompeted by naturally occurring rhizobia (Meade
et al., 1985). Partner choice is not only determined by the
abundance of the various bacteria but also depends on the
competitiveness for nodulation of these strains (Triplett and
Sadowsky, 1992; Laguerre et al., 2003). Therefore, understanding
mechanisms behind competitiveness for nodulation in pea may
lead to improved inoculation strategies.
The nodulation process has a high metabolic cost for both
rhizobial and legume partners (Phillips, 1980; Schulze et al., 1999;
Trainer and Charles, 2006; Voisin et al., 2007). The establishment
of the symbiotic interaction is also a complex evolutionary
process where the interests of the host and the bacteria are
not always aligned. There seem to be mechanisms allowing the
plant to monitor the nitrogen-fixation performance of symbiotic
bacteria and to sanction the inefficient strains (Simms and Taylor,
2002; Oono et al., 2011). However, poorly-fixing rhizobial strains
often gain advantage over beneficial strains despite offering poor
growth benefit to their plant host, indicating that additional
factors may influence fitness of symbiotic bacteria (Marco et al.,
2009; Sachs et al., 2010; Fujita et al., 2014). Domesticated pea and
faba bean crops tend to have fewer compatible symbionts than
their wild relatives (Mutch and Young, 2004). Whether modern
cultivars are less able to establish beneficial associations than
older landraces in these species, as was observed for soybean,
remains to be seen (Kiers et al., 2007).
The genetic basis of pea-Rlv partner choice when roots are
exposed to a mixture of compatible Rlv strains is far from being
fully understood. This study investigates the genetic variability
of partner choice in the pea-Rlv symbiosis. A pea core-collection
representative of the genetic and biogeographic diversity found
within the genus Pisum was inoculated with a mixture of
five diverse compatible Rlv strains. Differences in Rlv choice
according to pea diversity and selection history were investigated.
The relationship between nodulation competitiveness and
nitrogen fixation efficiency was evaluated. Consequences for
breeding and inoculation strategies for improving symbiotic
traits in the pea crop are discussed.
MATERIALS AND METHODS
Biological Material
One hundred and four Pisum accessions were selected from the
reference pea collection available at INRA Dijon (http://www.
thelegumeportal.net/), according to their diversities of genetic
or geographical origin and their variability in agronomic traits,
such as their cultivation status, end-use and sowing type (Burstin
et al., 2015). This 104-pea collection includes 12 wild or semi-
wild genotypes, 36 landraces, 12 inbred lines or germplasm, and
44 cultivars (Table S1). Accessions originate from as many as 36
countries, from known centers of diversity and domestication
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Bourion et al. Genetic Diversity of Pea-Rhizobium leguminosarum Partner Choice
(Middle East, Ethiopia, Afghanistan) or from areas where
domesticated peas were subsequently disseminated, in Southern
Africa, Asia, Europe and America. Among the wild, semi-wild or
landrace genotypes, two are P. fulvum accessions and nine are
P. sativum accessions identified as belonging to the subspecies
abyssinicum,elatius or humile. The panel of 44 cultivars is
representative of the variation in end-use, sowing type and other
characteristics of peas cultivated since the end of seventeenth
century.
The five selected Rlv strains were previously identified as
nodulating pea (Table S2) and have diverse geographical origins.
SA (P1NP2H) and SD (P1NP2K) are two strains originating from
France (Laguerre et al., 2003). SE (SL16) is a strain collected
in Algeria. SK (SpR) and SF (RifR) are spontaneous antibiotic
mutants, respectively of the reference strain 3841 originating
from England (Brewin et al., 1980, 1983), and of the strain
TOM originating from Turkey and known to be required for
nodulation by some Afghan peas (Winarno and Lie, 1979; Young
et al., 1982).
Pea Collection Genetic Structure Analyses
The 104 pea accessions were genotyped using the GenoPea
13.2 K SNP Array (Tayeh et al., 2015). A filtering was performed
to exclude highly heterozygous SNPs, which are not expected
given the high selfing rate in pea, and SNPs with a minor
allele frequency of <0.02. Following these steps, a set of 11,218
markers was used for population structure analyses. The genetic
structure of the sample was investigated using two methods:
(1) a model-based Bayesian clustering assignment algorithm
implemented in the software fastSTRUCTURE (Raj et al., 2014)
and (2) a discriminant analysis of principal components—
DAPC—a multivariate method which employs PCA to reduce
the number of correlated variables (SNP markers) to be analyzed
using a discriminant analysis implemented in the R package
Adegenet (Jombart et al., 2010). The fastSTRUCTURE analysis
was run for a number of clusters (K) ranging from 1 to 20 with
5 replicates per K-value and using the “simple prior” option
(Figure S1). To evaluate the repeatability of runs and check for
the absence of true multimodality, the program CLUMPP v.1.1.2
was run using the Greedy algorithm (Jakobsson and Rosenberg,
2007). The putative optimal number of clusters was assessed
from the likelihood profile and admixture plots were obtained
using a custom python script. The second method, DAPC, was
run without prior knowledge of groups. The optimal number
of clusters was thus assessed through sequential K-means and
model selection using the Bayesian information criterion. The
number of principal components was determined to be 2 through
maximization of the α-score measuring the difference between
the proportion of successful reassignment of the analysis and
values obtained using random groups.
Growth Conditions and Phenotyping
Plants were grown in a greenhouse under controlled temperature
(21/16◦C) in a 16/8-h day-night cycle and under a mean
photosynthetically active radiation of 250 µmol photons m−2
s−1furnished with high-pressure sodium lamps. Prior to sowing
in pots, the seeds of P. fulvum and of some P. sativum subsp.
elatius or humile accessions were scarified, and the seeds of all
the accessions sterilized in 10% bleach for 10 min and rinsed five
times in water. Seeds were sown on square 2-L pots previously
sterilized and filled with a 1:1 (v/v) mixture of previously
sterilized attapulgite and clay balls (2–6 mm diameter). Pots were
top-watered from day one until 8 days after sowing.
In a first experiment (E1), a three-block randomized design
was used, with two pots per accession in each block and three
seeds sown per pot. At sowing, each seed was inoculated with
1 mL (∼108cfu) of a rhizobial inoculum comprising an equal
proportion of the five Rlv strains. Eight days after sowing,
two of the three plantlets were kept per pot, always removing,
when possible (i.e., when three plants were present), the plant
from the same corner irrespective of its growth characteristics.
To allow both nodulation and sufficient plant growth to take
place (Moreau et al., 2008; Bourion et al., 2010), they were
supplied with a low nitrate content (0.625 mM) nutrient solution
(Table S3). Four weeks after sowing, all 1,248 plants kept were
harvested. For each accession, the nodules formed on roots were
counted on one of the two plants in one pot per block and
their total dry matter determined. Rhizobia were isolated for each
accession from a sample of 60 nodules randomly collected on the
two plants of the other pot of the block. After nodule surface
sterilization, each nodule was crushed and undifferentiated
bacteria cultivated on YM agar plates. Bacterial identities of
individual nodules were determined either by assaying antibiotic
resistance on YM medium supplemented with Rif (300 mg L−1)
for SF strain and with Sp and St (each at 300 mg L−1) for SK
strain and by PCR amplification using specific primers for SA,
SD and SE (Table S2). In most cases the bacteria present in each
nodule were clonal and we found little evidence for multiple
infection (mixed nodule) (<1%; data not shown). The frequency
of nodules containing each strain was then calculated for each
pea genotype, allowing estimation of the competitiveness of the
strains.
In a second experiment (E2), 18 pea accessions selected
from the 104 were mono-inoculated with each of the five Rlv
strains or, as a control, without any inoculation (NI). Each
mono-inoculation (or lack of inoculation) was performed in one
assigned bank not adjacent to the others, in order to prevent
cross-contamination. A four-block randomized design was used,
with one pot per accession in each block, and four seeds sown
per pot. At sowing, except for the NI control, each seed was
inoculated with a 1 mL cell suspension of one of the five Rlv
strains (∼107cfu). Eight days after sowing, two of the four
plantlets were harvested in each pot, always when possible
from the same two diagonally opposing corners. The absence
of nodules was confirmed in the plants from the NI bank. The
842 remaining plants were supplied with a nutrient solution
without any N until harvest time, 5 weeks after sowing (Table S3).
For each pea accession inoculated, all the nodules formed on
the two plants were counted, and a sample of 16 nodules was
collected over the four blocks. Nodule identity was checked using
the same method as in E1. The few cross-contaminated plants
were discarded and the shoot dry matter of the remaining plants
determined. Under mineral N-free conditions, symbiotic N
acquisition is generally the main limiting factor for plant growth
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Bourion et al. Genetic Diversity of Pea-Rhizobium leguminosarum Partner Choice
and the shoot dry matter determined for each legume-Rhizobium
association is indicative of its nitrogen fixation efficiency, as
illustrated in both the model legume Medicago truncatula and
pea (Laguerre et al., 2007, 2012; Moreau et al., 2008; Voisin
et al., 2010). In order to compare the efficiency of N fixation
while controlling for differences in growth potential between pea
accessions, we calculated a normalized shoot dry matter index.
For a given strain and pea accession, the index was calculated
by dividing the shoot dry matter obtained by the mean shoot
dry matter of this pea accession mono-inoculated with all five
strains (Heath and Tiffin, 2009). Likewise, a nodulation index was
calculated, as the nodule number of a pea accession inoculated
with a strain divided by the mean nodule number of this pea
accession.
RESULTS
Genetic Diversity of the Pea Collection and
of the Rlv Strains
Two complementary methods, DAPC and fastSTRUCTURE,
were used to assess the population genetic structure of the 104
accessions of the pea collection. After filtering, a set of 11,218
SNPs among the 13,204 of the GenoPea SNP Array (Tayeh
et al., 2015) were used for these analyses. The DAPC analysis
uncovered three genetic groups and all accessions except five had
a membership probability to one of them higher than 0.95 (Table
S1, Figure 1). There was a clear distinction between the three
groups according to the cultivation status and type of sowing of
FIGURE 1 | Genetic structure of the 104 accessions of the pea collection
using discriminant analysis on principal components (DAPC). After filtering, a
set of 11,218 SNPs was used for these analyses (see main text). The first
discriminant axis segregated wild vs. cultivated spring or winter peas. The wild
group comprises wild or semi-wild accessions and landraces from centers of
domestication. The second axis mainly discriminates spring and winter
cultivated peas. Note the ambiguous positions of three landraces or traditional
cultivars, “JI190” originating from Sudan, “Pisum sativum-Hibernicum JI1846”
from Egypt and “Capsicum” from Azerbaidjan and of two winter European
cultivars “Hativer” and “Cheyenne”.
the accessions. D1 was named “Wild group” as it comprised nine
of the 12 wild or semi-wild accessions plus 12 Pisum sativum
landraces from Abyssinia, Afghanistan and Asia (Table S1).
Among these landraces, “JI190,” originating from Sudan, had a
membership probability to D1 lower than 0.70. The D2 “Spring
group” comprised 64 accessions of which 48 of the 51 accessions
were spring sowing types. The D3 “Winter group” comprised 18
genotypes among which 15 were winter and three were spring
sowing types. Two of these accessions identified as spring sowing
types were sampled at an altitude of more than 2000 meters
(JI1844 and JI1431; JIC Pisum Collection database, https://www.
seedstor.ac.uk), supporting the cold tolerance characteristics of
D3. “Pisum sativum-Hibernicum JI1846” identified in the JIC
database as a spring sowing type was less related to the D3
group. Three other accessions, “Capsicum” of unknown sowing
type, and the two winter cultivars “Hativer” and “Cheyenne” also
displayed an ambiguous position between D2 and D3.
The fastSTRUCTURE analysis identified 10 different clusters
(K01–K10) which were interestingly found to further subdivide
the three DAPC groups (Figure S2). Based on the study of the
70 accessions with cluster membership probabilities higher than
80%, the cluster assignments were found to correlate with species
affiliation or geographic origin and breeding history (Table S1).
Of the three clusters subdividing the “Wild D1” group, K01
grouped the two P. sativum subsp. abyssinicum and of wild
accessions, the two P. fulvum and three P. sativum subsp. elatius
or humile, originating from the Middle East. K02 included three
of the four accessions from Afghanistan, a P. sativum subsp.
humile and all the three accessions from Nepal or India. K03
consisted of three P. sativum from Abyssinia (Ethiopia, Sudan)
or Libya. Within the D2 “Spring group,” K04, mainly consists of
landraces or fodder peas from Baltic States, Ukraine, Russia or
Sweden (“Torsdag,” registered in 1925). Garden peas have been
separately selected in France, Great Britain and the Netherlands
from the eighteenth century until the end of the 1960’s, which is
consistent with their separation into the three distinct clusters:
K05, with old French garden pea cultivars (including “Corne
de Bélier,” 1818); K06 with English garden peas (including
“Téléphone à rames,” 1878); and K07, with two Dutch accessions
and a P. sativum subsp. elatius (JI1703) whose origin is unknown.
K08, the fifth cluster in D2, grouped French spring dry pea
cultivars (including “Baccara,” the most cultivated during the
1990s). It is representative of the change in end-use, from garden
toward dry field peas for animal feed, which occurred in Europe
from the 1970’s. Within the D3 group, K09 and K10 gathered
respectively the winter European fodder peas and the French
winter pea cultivars (including “Frisson,” 1979).
Phylogenetic analyses based on symbiotic nod markers have
shown that the five strains used in this study (SA, SD, SE, SK, and
SF) are representative of a large diversity within the symbiovar
viciae (Figure S3).
Natural Variability in Pea Nodulation and
Pea-Rlv Partner Choice
In the first experiment E1, the 104 pea accessions were assessed
for their ability to form nodules and for their choice among
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Bourion et al. Genetic Diversity of Pea-Rhizobium leguminosarum Partner Choice
the five Rlv strains. Significant variation in nodule biomass and
number were observed among the 104 pea accessions when co-
inoculated with an equal mixture of the five Rlv strains (Table
S4). The lowest biomasses were observed in the wild accessions
in K01 (P. fulvum,P. sativum subsp. elatius or humile) or in
the fodder and dry pea cultivars belonging to K08, K09, and
K10, whereas the highest ones were obtained by landraces or
garden pea cultivars (K04, K05, or K07; Figure S4). A significant
positive correlation was observed between shoot dry matter and
both nodule dry matter and number (r2=0.76, p<0.0001 and
r2=0.47, p<0.0001 respectively). No significant effect of the
status of the pea accessions (wild, landrace, germplasm, breeding
line or cultivar) was observed in these relationships (Figure 2,
Table S5).
A large variability in the relative frequency of nodule
formation by the five Rlv strains was observed among the 104
pea accessions (Figure 3). With a mean frequency value of 67%
among the 104 accessions, globally SA was the most competitive
strain, far ahead of SD (14%), SK (13%), SF (5%), and SE
(2%) (Figure S5). However, variation between pea accessions was
found around these mean values, and differences were observed
according to their membership to DAPC groups (Figure 4A,
Table S6). The “Wild group” D1 presented a higher diversity in
Rlv choice than the groups D2 and D3 which included all the
pea cultivars. The relative frequency of SA varied much more
in D1 than in D2 or D3 in which it was always higher than
40%. SF was detected in members of D1 with a frequency up
to 97% and in none of the members of D2 or D3 except R038
(“Capsicum”). The maximum of the SK relative frequency was
higher in D1 than in D2 or D3. Differences were also observed
between the three clusters in the D1 group (Figure 4B, Table
S6). In K01, very few accessions had a strain preference (i.e.,
frequency higher than 80%). In K02, the relative frequencies
FIGURE 2 | Relationship between shoot dry matter and (A) nodule dry matter
or (B) nodule number per plant, for 104 pea accessions multi-inoculated with
a mixture of five Rlv strains (E1 experiment). Each point represents mean
values for one pea accession measured 4 weeks after sowing. Symbols are
different and colored according to cultivation status. Lines and equations
represent linear regression results (both P<0.001).
of SA and SF were highly variable, with a strain preference
either for SA or SF. In K03 all the accessions had a relative
frequency of SA higher than 91% and none was nodulated
by SF.
Correlation between Competitiveness and
Efficiency
To investigate whether competitiveness for nodulation was
related with nitrogen fixation efficiency, 18 pea accessions were
inoculated separately with each of the five Rlv strains, and
their shoot biomasses measured in absence of mineral N supply
(E2 experiment). The 18 accessions were selected from the
E1 experiment as displaying contrasted partner choice (Figure
S6A). They belonged either to the D1 or the D2 group and
were representative of the shoot and nodule biomass variations
encountered within the pea collection (Figure S6B, Table S1).
In almost all pea-Rlv combinations tested we could observe
nodulation, indicating that all five strains were able to form
nodules regardless of the plant genotype even though the number
of nodules varied drastically (Figure S7). The mean values of
shoot dry matter obtained for the five strains were correlated
to mean nodule numbers (r2=0.62, p<0.0001; Figure S8A).
Both pea genotype and Rlv strain were found to have significant
effects on nodulation and shoot dry matter of the 90 pea-Rlv
combinations, and the interaction between the two factors was
significant for both traits (Table S7, Figures S7, S9). Mean values
of shoot dry matter obtained for the 18 accessions over the five
strains were significantly and positively correlated with those
obtained in E1 (r2=0.51, p<0.001; Figure S8B), probably
because a large part of the variation is related to differences
in growth potential among the pea accessions. To overcome
this problem of growth potential differences, we calculated a
normalized index for both shoot dry matter and nodulation (see
Mat and Meth).
FIGURE 3 | Strain frequencies in the nodules of 104 pea accessions
multi-inoculated with a mixture of five Rlv strains (E1 experiment). Rhizobia
were isolated for each pea accession from a sample of 60 nodules randomly
collected on two plants 4 weeks after sowing.
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Bourion et al. Genetic Diversity of Pea-Rhizobium leguminosarum Partner Choice
FIGURE 4 | Strain frequencies in the nodules of pea accessions multi-inoculated with a mixture of five Rlv strains (E1 experiment), (A) within the 104 pea accessions
according to their membership to DAPC groups, (B) within the subset of pea accessions belonging to D1 according to their membership to K01, K02 or K03 clusters.
Crosses and bold lines respectively indicate mean frequency values and medians. Open circles represent outliers identified by accession numbers.
The nodulation indexes obtained with SA, SD, SE, and
SK were not correlated to the competitiveness for nodulation
of these strains, as evaluated in E1 (Figure 5, Table S8). As
such, the SE strain displayed an overall high ability to form
nodules but in all cases had low competitiveness, whereas SA
was the most competitive strain in multi-inoculation despite
its low nodulation ability. SF was the only strain for which a
correlation between the nodulation index and its competitiveness
could be detected (r2=0.79, p<0.000001). Indeed, SF
was the most competitive with three accessions belonging to
K02 (R098 and R029 from Afghanistan; R069 from Israel)
having the highest nodulation index, and not competitive at all
with the numerous accessions having a low nodulation index
with it.
Overall differences in shoot dry matter index were associated
with the Rlv strain (Figure 6). Globally, SF and SD promoted the
highest biomass production and SE was the least efficient in all
cases. Some differences in efficiency were observed depending
on the pea accession; SF being for instance the most efficient for
two pea accessions (R069, R098) and SA for the two accessions
belonging to K04 (R046, R043). However, competitiveness and
shoot dry matter index were found to be poorly correlated
(Figure 6, Table S9). Significant but dispersed correlations were
observed only for the two strains SF (r2=0.47, p=0.001) and
SA (r2=0.41, p=0.002). For both strains, the slope of the
regression line was lower than one indicating that increase in
shoot dry matter index was associated with moderate change in
competitiveness.
DISCUSSION
To our knowledge, this is the first time that the genetic diversity
for pea-Rlv partner choice has been investigated within a pea
collection representative of the variability within the genus Pisum
and co-inoculated with a mixture of diverse Rlv strains. Little is
known about the mechanisms underlying the competitiveness of
Rlv strains for pea nodulation and the selection effected by pea
genotypes faced with diverse compatible Rlv strains. Whether the
competitiveness for nodulation of pea-Rlv symbiotic association
is related to nitrogen fixation efficiency has been so far under-
investigated.
Nodulation Is Strongly Correlated to Shoot
Growth
Some differences in shoot dry matter were observed between
the pea accessions according to their cultivation status or
end-use, thus giving an overview of the modifications which
have occurred during domestication and subsequent breeding
history. Consistently, the landraces of P. sativum or P. s. subsp.
abyssinicum form larger plants than the P. fulvum accessions and
most of the other wild peas, and the low biomasses observed
in dry peas are consistent with the breeding evolution toward
field production of seeds for animal feeding. However, significant
positive correlations between shoot biomass and nodule biomass
or number were observed independently of species affiliation
and breeding history. A close correlation between symbiotic
organ development and plant growth has already been observed
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Bourion et al. Genetic Diversity of Pea-Rhizobium leguminosarum Partner Choice
FIGURE 5 | Relationship between nodulation index in mono-inoculation (E2
experiment) and strain competitiveness for nodulation in multi-inoculation (E1
experiment) for 18 contrasted pea accessions. Squares represent European
strains. Line and equation represent significant linear regression results
(P<0.001).
using samples exhibiting less genetic diversity within pea or M.
truncatula (Bourion et al., 2010; Voisin et al., 2010; Laguerre
et al., 2012). Pea and M. truncatula form indeterminate nodules,
following two sequential processes: nodule formation and mature
nodule expansion. Nodule formation results from early plant x
rhizobia interaction. It occurs under situations of whole plant N
limitation and is repressed by high N supply (Jeudy et al., 2010;
Voisin et al., 2010). Nodule expansion is also strongly dependent
on a systemic N-signaling regulation (Laguerre et al., 2012). In
our study (moderate nitrogen supply) shoot biomass was more
closely adjusted to nodule biomass than to nodule number. This
suggests that pea adjusts its symbiotic capacity to its N demand
by modulating C allocation for the expansion of nodules. Nodule
biomass is therefore more linked to late adjustment processes
than to nodule number.
Partner Choice Varies According to
Genetic Diversity
The “wild peas” group presented more variation with regard
to symbiotic partner choice than cultivated peas, in agreement
with previous observations (Lie, 1978; Lie et al., 1987). Within
the “wild” D1 group, the highest diversity in partner choice was
observed in the K01 cluster comprising the most primitive peas
(i.e., P. fulvum accessions and peas representative of the early
domestication events such as P. sativum subsp. abyssinicum and
P. sativum accessions of the Middle East genetic diversity center).
The K02 cluster was representative of peas from Afghanistan and
India, and comprised the landrace peas resistant to nodulation
with the European strains SA, SD, and SK, and highly specific
to nodulation with SF/TOM. All the accessions of K03 originate
from Abyssinia or Lybia, and display a high preference for
the SA strain. This tight association between a European
strain and African peas has not been previously reported. All
FIGURE 6 | Relationship between shoot dry matter index in mono-inoculation
(E2 experiment) and strain competitiveness for nodulation in multi-inoculation
(E1 experiment) for 18 pea accessions. Squares represent European strains.
Lines and equations represent significant linear regression results (both
P<0.05).
these observations suggest that the domestication process and
subsequent spread of peas from the Fertile Crescent may have
resulted in a loss of the ability to establish symbiosis with
some rhizobial strains. The cultivated pea groups (D2 and D3)
encompass a great variety of end-uses, sowing types (spring vs.
winter) and represent various steps in breeding history. Yet, no
differences in partner choice are apparent among them: they
essentially all choose predominantly SA. This does not support
the idea that breeding practices under increasingly N-rich soils
have led to a change in choice patterns between old and recent
cultivars in pea, as seen in soybean (Kiers et al., 2007).
The Nodulation Ability of a Strain Does Not
Predict Its Competitiveness for Nodulation
Experiments of mono- and multi-inoculation give
complementary information and new insights into the natural
variability of pea-Rlv partner choice. Our mono-inoculation
results indicated that SF (TOM) is required for the nodulation
of P. humile JI241 (R069) from Israel and for some Afghan
pea accessions (R098, R029), confirming previously reported
cases of host-controlled nodulation restriction (Lie, 1978;
Young and Matthews, 1982). We also found in agreement with
these authors that some other Afghan peas are only partially
resistant or even susceptible to European Rlv strains. We
even found further evidence for partially resistant accessions
in neighboring countries (in the Caucasus). We confirmed
the ability of SF/TOM, in a single-inoculum environment, to
nodulate a wide spectrum of pea accessions (Lie, 1981; Young
et al., 1982). However, we observed that SF, when inoculated
in a mixture with the other four Rlv strains, was competitive
only with the resistant accessions but not with the susceptible
ones. The inhibiting effect of European strains on nodulation
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Bourion et al. Genetic Diversity of Pea-Rhizobium leguminosarum Partner Choice
by SF/TOM, named competitive nodulation blocking (cnb), has
been well documented on the resistant pea line “cv. Afghanistan”
(Winarno and Lie, 1979; Dowling et al., 1989; Hogg et al.,
2002). Evidence was provided that the high levels of Nod
factors produced by these cnb+European strains account for
their nodulation blocking of “cv. Afghanistan” (Hogg et al.,
2002). Our observation of the inhibition by four Rlv strains of
nodulation by SF of susceptible peas does indicate that another
competitiveness mechanism exists besides the cnb+effect.
Moreover, no correlation was observed for the four strains
other than SF/TOM between their nodulation ability and their
competitiveness for nodulation when inoculated in a mixture.
All these results highlight that nodulation competitiveness
is controlled by multiple genetic factors from both pea and
rhizobia.
Other LysM-RLKs pea genes besides SYM2 have been shown
to be required for Nod factor perception (Madsen et al., 2003;
Zhukov et al., 2008). Whether the variation in Nod factor
perception together with the strain-dependent variation in Nod
factor production is responsible for changes in competitiveness
of various compatible pea-Rlv associations merits investigation.
Furthermore, little is known about the role in pea-Rlv
symbiosis of the control mechanisms revealed in model legumes
and involving either rhizobial cell surface polysaccharides or
secretion of rhizobial effector proteins (Masson-Boivin et al.,
2009; Downie, 2010; Kawaharada et al., 2015; Malkov et al., 2016).
Competitiveness for Nodulation and
Nitrogen Fixation Efficiency Are Not
Correlated
It is conceivable that competitiveness for nodulation and
efficiency of symbiotic association have been co-selected, favoring
the best growing symbiotic plants. However, we found very
little evidence that competitiveness is related to nitrogen fixation
efficiency. A weak (albeit significant) relationship between strain
competitiveness (multi-inoculation) and strain efficiency (mono-
inoculated plants) was only found for two strains, SA and SF. This
might arise mainly from differences between accessions in their
level of resistance to nodulation by European strains and their
specificity for SF. Thus, a high competitiveness of a given strain
does not ensure high nitrogen-fixing efficiency and high biomass
production for the plant. The several studies investigating the
relationship between competitiveness and efficiency of symbiotic
association have provided contrasted results according to the
stage of observation. The poor relationship we observed is in
agreement with the observation that Fix−mutants of R. meliloti
do not significantly differ in nodulation competitiveness with
alfalfa (estimated 10 days after inoculation) from their Fix+
parental strains (Amarger, 1981). More recently we showed
in M. truncatula/Sinorhizobium that plant N status does not
impact initial partner choice for nodule formation but results
in preferential later expansion of the nodules formed with the
most efficient strains (Laguerre et al., 2012). Accordingly, a
long-term preference for efficient strains of indigenous rhizobia
was observed in 2 months-old alfalfa plants selected for high
levels of nitrogen fixation (Hardarson et al., 1982). Preference
for efficient associations requires late symbiotic interactions as
a function of plant N demand, resulting in a strong allocation
of metabolites by the plant to the nitrogen-fixing bacteroids.
Conversely, competitiveness for nodulation is most probably
determined during early plant-bacterial interactions and/or the
bacterial colonization of the symbiotic organ but may not be
directly related to the amount of N supplied through symbiosis.
Our study highlighted that competitiveness for nodulation and
nitrogen fixation efficiency must both be considered as selection
criteria for improving pea crop production.
CONCLUSION
Our work underlines the importance of viewing legumes as being
exposed to several compatible symbiotic rhizobia rather than
to a single strain. Mono-inoculation experiments are useful for
evaluating the ability of a strain to form efficient symbiosis with
a given host, but they fail to determine the ability of this strain
to compete with other strains in a mixture, which is the situation
commonly encountered in the field. A successful inoculant must
not only provide enhanced symbiotic nitrogen fixation but be
competitive for nodulation in the presence of a large population
of indigenous rhizobia. Nodulation competitiveness is controlled
by genetic factors from both the plant and the bacteria. Further
experiments are in progress to decipher the genetic basis of
this trait. Genome Wide Association Studies, involving larger
pea and Rlv collections, increased genomic resources and higher
throughput phenotyping technologies than those used in this
study, may fulfill this challenge. Such knowledge of the key
genetic factors involved will be essential for developing new pea
varieties and Rlv inoculants for improved pea-Rlv symbiosis.
AUTHOR CONTRIBUTIONS
VB and ML co-coordinated the overall study that was also
initiated by Gisèle Laguerre. GD contributed to the conception
of the study and the design of the pea collection. JB contributed
to the conception of the study and coordinated the genotyping of
the pea collection. MS performed pea collection genetic structure
analyses. VB contributed to the design of the pea collection,
coordinated and participated in greenhouse experiments and
plant phenotypic data acquisition, performed plant phenotypic
data analyses and interpretation. VA and KH-G participated in
greenhouse experiments and contributed to phenotypic plant
data acquisition. MC-M and CD provided seeds and information
on the pea collection, and contributed to phenotypic data
acquisition. BB and MP performed phylogenic analysis of the
Rlv bacteria. BB, KH-G, ML, and PT collected nodules and
performed analyses of bacterial occupancy. DV contributed to
statistical analysis of the data. VB and ML wrote the manuscript
which was revised and accepted by all authors.
ACKNOWLEDGMENTS
The authors greatly acknowledge Philip Poole (JIC, UK) for
providing the TOM and 3841 strains. The authors thank
Frontiers in Plant Science | www.frontiersin.org 8January 2018 | Volume 8 | Article 2249

Bourion et al. Genetic Diversity of Pea-Rhizobium leguminosarum Partner Choice
Karine Palavioux, Franck Zenk, Noureddine El Mjiyad, and
Damien Gironde (UMR1347, Dijon) for their technical support
in the greenhouse experiments; Nicolas Carlier, Laetitia Dalla
Corte, Henri de Larambergue, Hervé Houtin, Anthony Klein,
Chantal Martin, Jean-Bernard Magnin-Robert, and Céline Rond
(UMR1347, Dijon) for their help in plant phenotyping; Jean-
Michel Retailleau (GEVES, Brion), Peter Spencer-Phillips (UWE,
Bristol), Marie-Laure Pilet-Nayel (UMR IGEPP, Rennes), and
Dominique Millot (UMR1347, Dijon) for providing information
about several pea accessions; Fabrice Dessaint (UMR1347, Dijon)
for its help concerning the statistical analyses in R. This work
was supported by the French ANR GENOPEA and the “INRA-
BAP projet scientifique” SYMBIOPEA programs. The authors
thank Antoine Le Quéré (LSTM, Montpellier) and Richard
Thompson (UMR1347, Dijon) for their critical reading of the
manuscript.
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found
online at: https://www.frontiersin.org/articles/10.3389/fpls.2017.
02249/full#supplementary-material
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Frontiers in Plant Science | www.frontiersin.org 10 January 2018 | Volume 8 | Article 2249