Symbiosis

DOI 10.1007/s13199-017-0475-6

Symbiotic performance, nitrogen flux and growth

of lima bean (Phaseolus lunatus L.) varieties inoculated
with different indigenous strains of rhizobia
Vicente Paulo da Costa Neto 1 & Janaina Barros Siqueira Mendes 1 &
Ademir Sérgio Ferreira de Araújo 2 & Francisco de Alcântara Neto 3 &
Aurenivia Bonifacio 4 & Artenisa Cerqueira Rodrigues 5

Received: 8 October 2016 / Accepted: 27 January 2017

# Springer Science+Business Media Dordrecht 2017

Abstract Legumes, such as lima bean, can form symbiotic
relationships with rhizobia and can therefore grow in nitrogen-
poor soils. Lima bean varieties in symbiosis with indigenous
rhizobia were evaluated for their symbiotic performance over
three harvest periods. Four indigenous rhizobial strains
(ISOL-16, ISOL-18, ISOL-19 or ISOL-35) were isolated from
soil samples in lima bean fields and used separately to inocu-
late two lima bean varieties (‘boca de moça’ and ‘branca’).
Uninoculated, unfertilized plants were used as controls, and
uninoculated, nitrogen-supplied plants were used as the nitro-
gen control. All inoculated plants and the uninoculated control
were cultivated using nitrogen-free nutrient solutions in the
greenhouse. As expected, the uninoculated plants did not de-
velop nodules on their root systems and were inferior in all
evaluated parameters. The lima bean nodulated by
Bradyrhizobium exhibited growth variables, nodules parame-
ters, and nitrogen flux values superior to those of plants inoc-
ulated with Rhizobium. The highest total chlorophyll values
were recorded in lima bean inoculated with Bradyrhizobium,
confirming that these plants had the greenest leaves and likely
have superior photosynthetic efficiency – a hypothesis sup-
ported by the greater growth exhibited by these plants.

Highlights
• Inoculation with Bradyrhizobium strains induces better N-flux and
growth in lima bean
• N-fix efficiency was superior in lima bean nodulated by
Bradyrhizobium strains
• Bradyrhizobium is most efficient in establishing a successful symbiotic
relationship with lima bean
Electronic supplementary material The online version of this article
(doi:10.1007/s13199-017-0475-6) contains supplementary material,
which is available to authorized users.

* Artenisa Cerqueira Rodrigues 1

Plant Production Post-Graduate Program, Federal University of
artenisacerqueira@hotmail.com Piaui, Teresina, PI, Brazil

Vicente Paulo da Costa Neto 2

Department of Agricultural Engineering and Soil Science,
costanetovp@gmail.com Laboratory of Soil Quality, Federal University of Piaui, Teresina, PI,
Brazil
Janaina Barros Siqueira Mendes
janabsmendes@gmail.com 3
Department of Plant Science, Federal University of Piaui,
Ademir Sérgio Ferreira de Araújo Teresina, PI, Brazil
asfaruaj@yahoo.com.br 4
Department of Biology, Laboratory of Plant Physiology, Federal
Francisco de Alcântara Neto University of Piaui, Teresina, PI, Brazil
fneto@ufpi.edu.br 5
Department of Agricultural Engineering and Soil Science,
Aurenivia Bonifacio Laboratory of Soil Microbiology, Federal University of Piaui, CEP,
bonifacio.a@live.com Teresina, PI 64049-550, Brazil

Nitrogen fixation efficiency was superior in lima bean
nodulated by Bradyrhizobium, indicating that this microbe
possesses a greater ability to fix nitrogen and provide it con-
tinuously to the host plant. The symbiosis between lima bean
and Bradyrhizobium sp. ISOL-18 displayed the best values
with respect to carbon and nitrogen flow. We conclude that
Bradyrhizobium is the most effective at establishing an effi-
cient and successful symbiotic relationship with lima bean and
emphasize the potential value of Bradyrhizobium strains as
inoculants in lima bean cultivation.
Keywords Nitrogen fixation efficiency . Diazotrophs .
Growth
1 Introduction
Nitrogen is an essential nutrient that is directly involved in
plant growth, and is , limiting in many soils (Andrews and
Lea 2013). Biological nitrogen fixation (BNF) is an important
nitrogen supply process that is performed by nitrogen-fixing
bacteria. These diazotrophs convert atmospheric dinitrogen
(N2) to forms that are utilizable by plants (ammonium), thus
improving the plant’s carbon fixation and transpiration rates
(Olivares et al. 2013; Figueiredo et al. 2016). Diazotrophs can
fix nitrogen as free-living microbes, but some establish sym-
biotic relationships with leguminous plant species and induce
the formation of root nodules, which are lateral root structures
connected to the host plant’s vascular system that create a
favorable environment for BNF (Peix et al. 2015). Root-
nodule bacteria, or simply rhizobia, are increase legume yield
(Servín-Garcidueñas et al. 2014), and they contribute to envi-
ronmentally safe agriculture (Figueiredo et al. 2013).
Biological nitrogen fixation appears to be a more sustain-
able technology that can reduce undesirable effects of exces-
sive use of chemical fertilizers (Olivares et al. 2013;
Figueiredo et al. 2016). Legumes are of great biological and
agricultural importance (Andrews and Lea 2013; Figueiredo
et al. 2013). Among legumes, the Phaseolus genus is very
important as a potential host for strains of rhizobia (Lira
Junior et al. 2015). This genus is one of the best-studied le-
gumes (Mora et al. 2014; Servín-Garcidueñas et al. 2014).
Among the species in the Phaseolus genus, P. vulgaris and
P. lunatus are the most important food legumes (Li et al.
2015). P. lunatus, commonly known as lima bean, is widely
cultivated by small farmers in northeast Brazil, a region with
nitrogen-poor soils (Lopes et al. 2015).
The lima bean is considered to be a host with limiting
microsymbiont specificity. It has been reported that
Bradyrhizobium species are their natural root-nodule symbi-
onts (López-López et al. 2013) of this plant and B. icense,
B. paxllaeri and B. yuanmingense have been documented as
symbionts of lima bean in its native habitat in Peru (López-
Costa Neto V. et al.
López et al. 2013; Duran et al. 2014; Matsubara and Zúñiga-
Dávila 2015). In Brazil, various Bradyrhizobium strains have
been identified in nodules of domesticated lima bean varieties
(Santos et al. 2011; Araujo et al. 2015). Some rhizobial strains
display a narrow host specificity, but others can form symbi-
oses with various legumes with varying efficacy, i.e., they are
less selective (Mora et al. 2014). Indeed, Rhizobium and
Sinorhizobium bacteria are reported as symbionts of lima bean
in Central and South America. However, the level of success-
ful inoculation by these symbionts was very low (Santos et al.
2011; Ormeño-Orrillo et al. 2012; Araujo et al. 2015).
Studies focusing on selecting potential symbionts for dif-
ferent Phaseolus species, particularly lima bean, are impor-
tant. They can establish which root-nodule bacteria are best
able to increase legume crop growth and yields (Servín-
Garcidueñas et al. 2014). The interest in lima beans and its
symbiotic relationship with strains of rhizobia has been grow-
ing in recent years due to the great economic and social im-
portance of this crop worldwide, and particularly in Brazil
(Araujo et al. 2015; Lopes et al. 2015). We hypothesized that
only effective rhizobia-plant symbioses would improve
growth in lima beans. In this context, indigenous rhizobia
from lima bean fields were evaluated.
2 Materials and methods
2.1 Isolation of indigenous rhizobia nodulating lima bean
The indigenous rhizobia isolates used in this study were obtain-
ed from root nodules of lima bean variety UFPI-491 trap plants
grown in plastic bags filled with non-sterile soil under green-
house conditions. The soil was collected from two fields with a
history of lima bean cultivation, and the rhizobia strains were
isolated using the procedure described by Araujo et al. (2015).
Based on the similarity of partial 16S rRNA gene sequences, 14
indigenous rhizobial strains were identified (Araujo et al. 2015),
and of these strains, four were selected for this study: one
Rhizobium strain (ISOL-16) and three Bradyrhizobium strains
(ISOL-18, ISOL-19, and ISOL-35). These isolates were chosen
based on the findings of Araujo et al. (2015).
2.2 Rhizobial strains and culture media
The indigenous rhizobial strains were purified in yeast
mannitol agar (YMA) medium using 0.25% (w/v) Congo
red as an indicator, and after purification, the indigenous
rhizobial strains were cultured in tubes with YMA medi-
um without the indicator. For inoculant preparation, the
indigenous rhizobial strains that had been individually
isolated in 250-mL Erlenmeyer flasks containing liquid
yeast mannitol (YM) medium were incubated in a rotator
shaker at 220 rpm (28 °C) for 120 h.
Symbiotic performance, nitrogen flux and growth of lima bean
2.3 Source and disinfection of lima bean seeds
Seeds of the two lima bean varieties (‘boca de moça’ and
‘branca’) were obtained from Brazilian commercial stores and
selected based on their size and physical integrity. The lima bean
seeds were disinfected using 70% (v/v) ethanol for 30 s and 2%
(v/v) sodium hypochlorite for 60 s. Subsequently, these seeds
were rinsed seven times with sterile distilled water, placed on
autoclaved paper towels and then dried in a flow chamber.
2.4 Greenhouse experiment
The experiment was conducted in a greenhouse at the
Department of Agricultural Engineering and Soil Science
(UFPI, Piaui, Brazil) within a temperature range of 28–
37 °C, 50–70% relative humidity and 1200-μmol m−2 s−1 of
photosynthetically active radiation. The lima bean varieties
‘boca de moça’ and ‘branca’ were inoculated with indigenous
rhizobia and simultaneously sown in Leonard jars containing
washed (pH 6.5) and autoclaved (120 °C; 101 kPa; 1 h) sand
in the tops. In total, 1.0 mL of YM medium with indigenous
rhizobia at 108 CFU mL−1 was added to each lima bean seed.
Uninoculated plants without additional nitrogen supply were
used as control, while uninoculated nitrogen-supplied plants
were used as a nitrogen control. After thinning the lima bean
seedlings at seven days, two plants were retained in each jar
(experimental unit). During the experimental period, the
plants were capillary irrigated with nitrogen-free Hoagland
and Arnon’s nutritive solution (Hoagland and Arnon 1950)
modified by Silveira et al. (1998a). For nitrogen control, the
sand in the top of the Leonard jars was moistened with the
ammonium sulfate (0.0084 g) diluted in sterile distilled water
at seven days until the end of the experiment.
2.5 Measurements
The plants were collected at three sampling times: (1)
flowering, i.e., the period of highest nitrogen fixation (37 days
after germination); (2) pod setting, i.e., the period of pod emis-
sion or stable nitrogen fixation at high rates (50 days); and (3)
pod filling, i.e., the period of declining nitrogen fixation (63 days).
The sampling times were chosen based on previously published
results (Matos 1991; Silveira et al. 1998b). Throughout the ex-
perimental period, the heights of the lima bean varieties were
measured, and the data were used to calculate the absolute
growth rate. At each sampling time, the stem diameter and root
length were measured, and the plant material was collected and
partitioned into shoots, roots and nodules. The number of nod-
ules was counted manually, and the dry weight of the shoots,
roots and nodules was determined after the samples had been
dried in a forced aeration oven at 65 °C to a constant weight.
Total plant dry weight was used to calculate the relative growth
rate according to Evans (1972). The nitrogen contents of shoots
and nodules were determined by Kjeldahl method as described
by Bremner (1965). Based on the above data, specific nodule
weight, specific nodulation, accumulated nitrogen, nitrogen con-
tent per shoot dry weight and nitrogen fixation efficiency were
calculated (Gulden and Vessey 1998). In each collection period,
total chlorophyll was determined using a portable electronic chlo-
rophyll meter (ClorofiLOG® CFL1030, Falker, Porto Alegre,
Brazil).
2.6 Experimental design and statistical analysis
The experiment employed a randomized block design with
four replications and a 4 × 3 × 2 + 2 factorial treatment ar-
rangement. The treatments included four indigenous rhizobia
strains, three harvest periods and two lima bean varieties with
two controls (uninoculated control without nitrogen, and un-
inoculated nitrogen-supplied control). Data were tested for
normality using the Shapiro-Wilk test (P < 0.05). Analysis
of variance (ANOVA) was performed on the experimental
data, and when the F test was significant, differences in mean
pairs were examined by Tukey’s test (P < 0.05). All statistical
analyses were performed with ASSISTAT software version
7.7 beta (Silva and Azevedo 2016).
3 Results and discussion
A common problem in the cultivation of legumes, such as
lima bean, is the low efficiency of the indigenous rhizobia
strains, so there is a need to search for more efficient
nitrogen-fixing inoculant strains for these species
(Mohammadi et al. 2012). Rhizobium and Bradyrhizobium
species induce beneficial effects in legumes mainly due to
their capacity to perform biological nitrogen fixation
(Lisitskaya and Trosheva 2013; Figueiredo et al. 2016). In this
study, one indigenous Rhizobium and three indigenous
Bradyrhizobium strains were selected to form symbiotic pairs
with the ‘boca de moça’ or ‘branca’ lima bean varieties, and
these different host-rhizobia combinations improved plant
growth to varying degrees and exhibited different levels of
symbiotic effectiveness. Lima bean inoculated with
Bradyrhizobium strains exhibited significant increases in ab-
solute and relative growth rates compared to the Rhizobium-
inoculated plants or the uninoculated plants (Fig. 1).
The absolute and relative growth rates reflect the rate of
plant growth in response to the treatments; these variables are
influenced by carbon fixation from photosynthetic processes
and by nutrient uptake, mainly nitrogen, by the roots
(Kirschbaum 2011; Lobato et al. 2013). In successful
rhizobia-legume symbiosis, rhizobia provide nitrogen and
stimulate photosynthesis in the plant host, resulting in a sig-
nificant increase in plant growth (Figueiredo et al. 2013; Lira
Junior et al. 2015). Indeed, it was observed in this study that
 Costa Neto V. et al.

a 3.5
b 35

rate (mg g-1 day-1)

Absolute growth
aA

Relative growth
aA aB bA

rate (cm day-1)

2.8 28 bB bB aB aB
aC bC aC
aC bA bA bD
2.1 aB 21 aD aE
bD bC
1.4 bE aD 14
bF aE
0.7 7
0.0 0
Boca de moça Branca Boca de moça Branca
Rhizobium sp. ISOL-16 Bradyrhizobium sp. ISOL-19 Nitrogen control
Bradyrhizobium sp. ISOL-18 Bradyrhizobium sp. ISOL-35 Absolute control

Fig. 1 Absolute (a) and relative (b) growth rates of ‘boca de moça’ and
‘branca’ lima bean varieties separately inoculated with four indigenous
rhizobia: Rhizobium sp. strain ISOL-16 or Bradyrhizobium sp. strain
ISOL-18, ISOL-19 or ISOL-35. Controls were uninoculated nitrogen-
supplied plants (nitrogen control) and uninoculated plants without
nitrogen supply (control). Different uppercase letters represent
significant differences among treatments in each variety, while different
lowercase letters indicate significant differences between the two varieties
by Tukey’s test (P < 0.05)

the ‘boca de moça’ and ‘branca’ lima bean varieties nodulated
by Bradyrhizobium sp. ISOL-18 had growth rates that were
higher compared with the other treatments. They were 35%
and 24% higher, respectively, than that of the nitrogen control
(Fig. 1b). It is probable that rhizobia inoculation enhances cell
elongation and dry weight accumulation in lima bean in re-
sponse to the increase in photosynthesis and the translocation
of photosynthate from source tissues (Kirschbaum 2011).
The translocation of photosynthate occurs via the phloem and
is necessary to maintain plant growth (Liu et al. 2012). Stem
diameter directly reflects photosynthate translocation, as thicker
stems enable more efficient transport (Kirschbaum 2011;
Lemoine et al. 2013). In this study, the inoculation of lima bean
varieties with Bradyrhizobium resulted in the largest stem diam-
eter (Supplemental Fig. S1) and also positively affected other
plant growth parameters, such as root length (Supplemental
Fig. S2) and dry weight of shoot and roots (Fig. 2).
Interestingly, lima bean varieties inoculated with
Bradyrhizobium sp. ISOL-18 exhibited a continuous increase in
shoot and root dry weight during the experimental period (Fig. 2).
Nitrogen fixed by rhizobia promotes plant mineral nutrition and
better nodule, root and shoot organ growth (Richardson et al.
2011; Lisitskaya and Trosheva 2013; Figueiredo et al. 2016).
Nodule development is an energetically expensive process,
so it must be finely controlled to optimize host plant growth
once the legume-rhizobia symbiosis is established (Tatsukami

Lima bean var. boca de moça

a Shoot dry weight b Shoot dry weight c Shoot dry weight
5 5 5 aA
4 4 bA 4
aC bB
3 bA bC aB 3 aD bC aB bD 3 aD
Dry weight
(g plant-1)

aD aD aE aE
2 2 bE 2 aF
1 1 1
0 0 0
-1 bD -1 bD
-1 bE
-2 bC bB aB aB -2 bC bB bA bB aB -2 aD
bA bB bB aC
-3 -3 -3 aA
Root dry weight Root dry weight Root dry weight
Lima bean var. branca
d Shoot dry weight e Shoot dry weight f Shoot dry weight
5 5 5
bA
4 aA 4 aA 4 aB
aC
3 3 3
Dry weight

aB aD aE aF
(g plant-1)

aB bC aC bE bD aC aF
2 bD bE 2 2
1 1 1
0 0 0
-1 aF -1 aE -1 aE
-2 aE aB aA aD bC -2 aD aC bB -2 aD aC aC
aA aA aA aB
-3 Root dry weight -3 Root dry weight
-3 Root dry weight
Rhizobium sp. ISOL-16 Bradyrhizobium sp. ISOL-19 Nitrogen control
Bradyrhizobium sp. ISOL-18 Bradyrhizobium sp. ISOL-35 Absolute control
Fig. 2 Shoot and root dry weight of ‘boca de moça’ (a, b and c ) and each harvest period, different uppercase letters represent significant dif-
‘branca’ (d, e and f) lima bean varieties separately inoculated with four ferences among treatments in each variety whereas different lowercase
indigenous rhizobia, Rhizobium sp. strain ISOL-16 or Bradyrhizobium letters indicate significant differences between the two varieties (boca de
sp. strain ISOL-18, ISOL-19 or ISOL-35, at flowering, pod setting or pod moça and branca). Statistical analysis was performed separately in each
filling. Controls were uninoculated nitrogen-supplied plants (nitrogen tissue. All mean comparisons were performed using Tukey’s test
control) and uninoculated plants without nitrogen supply (control). In (P < 0.05)
Symbiotic performance, nitrogen flux and growth of lima bean

and Ueda 2016). Although a sink for photoassimilates, nod-
ules provide greater amounts of rhizobia-fixed nitrogen to the
host plant and, therefore, nodule number is often correlated
with an effective legume-rhizobia symbiosis (Mortier et al.
2012; Figueiredo et al. 2016). Peláez-Vico et al. (2016) af-
firms that the nodules formation is a complex process divided
in two different developmental steps: bacterial infection initi-
ated in the epidermis and nodule organogenesis that takes
place in the root cortex. Lira Junior et al. (2015) reinforce that
nodules may be formed even when occurs an ineffective ex-
change of chemical signals between plants and rhizobia, and,
in this situation, or rhizobia do not enter and do not occupies
the nodule or when they enter simply do not fix nitrogen.
It was observed that Bradyrhizobium strains were able to
form nodules in taproots and lateral roots of lima bean, while
that nodule from Rhizobium sp. ISOL-16 were observed only
on lateral roots. Although Rhizobium and Bradyrhizobium
strains are able to form nodules lima bean roots,
Bradyrhizobium strains exhibited the highest nodules number
(Fig. 3). For example, at flowering, lima bean var. ‘boca de
moça’ and ‘branca’ inoculated with Bradyrhizobium sp.
ISOL-18 had approximately 260 and 560 nodules plant−1,
respectively; while that Rhizobium sp. ISOL-16 yielded only
34 and 30 nodules plant−1, respectively, in the same period.
Lima bean nodulated by Bradyrhizobium sp. UFLA 03–144
and grown in axenic conditions displays higher nodules num-
ber (506 nodules plant−1) at 45 days after sowing (Costa et al.
2017). Although number and mass of nodules are good indic-
ative of nodulation, nodule mass is more useful in evaluating
nodulation since it has a better correlation with symbiotic
performance (Döbereiner 1966). In this study, highest nodules
dry weight was also registered in lima bean inoculated with
Bradyrhizobium, particularly when strain ISOL-18 of
Bradyrhizobium was used (Fig. 3c, d). The nodules dry weight
varying between 0.5 to 0.7 g plant−1 in lima bean var. UFPI-
468 inoculated with native rhizobia strains (subsequently
identified by Araujo et al. 2015 as Bradyrhizobium strains)
was recorded by Antunes et al. (2011).
Although Rhizobium form nodules in lima bean roots, lima
bean varieties nodulated by Bradyrhizobium strains exhibits

a Lima bean var. boca de moça 640

b Lima bean var. branca
640 aA
Nodules number
Nodules number

(nodule jar-1)
(nodule jar-1)

480 480 aB
320 bA aA 320 bA
aB aC
bB
160 bC bB bC 160 aC
aD aA aB aD aD aC aA aB
bD bC NR NR
0 0
Flowering Pod setting Pod filling Flowering Pod setting Pod filling
c Lima bean var. boca de moça 0,4
0.4
d Lima bean var. branca
0,4
0.4
weight (g jar-1)
weight (g jar-1)

aA aA
Nodule dry
Nodule dry

0.3
0,3 bA 0.3
0,3 aB bA
aC
0,2
0.2 bC bB aB
0,2
0.2 aB aC aA
aD aD bC aC bB aA aD aD bC bB
0,1
0.1 0,1
0.1
NR NR
0.0
0,0 0.0
0,0
Flowering Pod setting Pod filling Flowering Pod setting Pod filling
e f
Specific nodule weight
Specific nodule weight

(mg nodule g-1 RDW)

(mg nodule g-1 RDW)

Lima bean var. boca de moça 240

Lima bean var. branca
240 aA
bA aA aB
180 180
bB
120 aC aB
120 bA
bD aC bC aB bB
aC bC aB aB aA aC aA bA
60 60 aB
NR NR
0 0
Flowering Pod setting Pod filling Flowering Pod setting Pod filling
g Lima bean var. boca de moça h Lima bean var. branca
Specific nodulation
Specific nodulation

360 360
(nodule g-1 RDW)
(nodule g-1 RDW)

aA
270 270 aB
bA
aA 180 aC
180
aB bA
bB bB aC
90 bC aC 90
aD bD bC NR aA bB bD aD aC bA aB
NR
0 0
Flowering Pod setting Pod filling Flowering Pod setting Pod filling
Rhizobium sp. ISOL-16 Bradyrhizobium sp. ISOL-18 Bradyrhizobium sp. ISOL-19 Bradyrhizobium sp. ISOL-35

Fig. 3 Number of nodules (a and b), nodule dry weight (c and d),
specific nodule weight (e and f), and specific nodulation (g and h) of
the ‘boca de moça’ and ‘branca’ lima bean varieties separately
inoculated with four indigenous rhizobial strains, Rhizobium sp. strain
ISOL-16 or Bradyrhizobium sp. strain ISOL-18, ISOL-19 or ISOL-35,
at flowering, pod setting or pod filling. In each harvest period, different
uppercase letters represent significant differences among treatments in
each variety whereas different lowercase letters indicate significant
differences between the two varieties (boca de moça and branca).
NR = No record of viable nodules. RDW = root dry weight. All mean
comparisons were performed using Tukey’s test (P < 0.05)
 Costa Neto V. et al.

highest nodules number, nodules dry weight, specific nodule a Lima bean var. boca de moça

N-fixation efficiency
600

(mg N g-1 NDW)

aA
weight and specific nodulation (Fig. 3) and this results indicates aB aA
450 aB
that this microsymbiont is more efficiently in establish symbio- aC
bA aB aD aC
sis with lima bean. It is notable that the symbiotic pair formed by 300 aD aC
lima bean and Bradyrhizobium sp. ISOL-18 displayed large and 150
active nodules that helped the plant maintain higher growth 0
NR
rates. This symbiotic pair displayed better plant growth and Flowering Pod setting Pod filling
nodule-related parameters compared to the other symbiotic b

N-fixation efficiency
600 Lima bean var. branca
pairs, thus making the plant-rhizobia symbiosis more efficient

(mg N g-1 NDW)

bA
(Figueiredo et al. 2013). The higher specific nodulation exhib- 450 bB bA bB
aA bC
ited by lima bean varieties inoculated with Bradyrhizobium was 300 bB bD
bC
superior than those of plants nodulated with Rhizobium in all bC
150 bD
harvest periods (Fig. 3g, h). Specific nodulation values were NR
gradually reduced during the experiment, and the lowest values 0
Flowering Pod setting Pod filling
were recorded during pod filling, the period when there is a
Rhizobium sp. ISOL-16 Bradyrhizobium sp. ISOL-19
smaller number of viable nodules (Fig. 3).
Bradyrhizobium sp. ISOL-18 Bradyrhizobium sp. ISOL-35
The better nodule-related parameters displayed by lima
Fig. 5 Nitrogen fixation efficiency in ‘boca de moça’ (a) and ‘branca’ (b)
bean varieties inoculated with Bradyrhizobium, mainly the lima bean varieties separately inoculated with four indigenous rhizobia,
ISOL-18 strain, indicate a better flow of photosynthetic car- Rhizobium sp. strain ISOL-16 or Bradyrhizobium sp. strain ISOL-18,
bon, which supplies the energy required for nodule organo- ISOL-19 or ISOL-35, at flowering, pod setting or pod filling. In each
genesis (Tatsukami and Ueda 2016). According to Mortier harvest period, different uppercase letters represent significant differences
among treatments in each variety whereas different lowercase letters in-
et al. (2012), the carbon supply is a signal that affects nodu- dicate significant differences between the two varieties (boca de moça and
lation and determines the efficiency of biological nitrogen branca). NR = No record of viable nodules. NDW = nodule dry weight.
fixation. The fact that specific nodulation is lower in lima bean All mean comparisons were performed using Tukey’s test (P < 0.05)
nodulated by Rhizobium sp. ISOL-16, combined with the in-
ferior plant growth parameters, indicates that the nodules in- related to the flow of nitrogen from rhizobia to the plant, i.e.,
duced by this strain are less efficient in symbiotic nitrogen accumulated nitrogen (Fig. 4) and the nitrogen content
fixation (see Figs. 1 and 2). Furthermore, the parameters (Supplemental Fig. S3) reinforce this idea.

a Lima bean var. boca de moça

180
aA
Accumulated N
(mg N SDW-1)

135 aA
90 bA
aB aB aB aB aB aB
45 aD aC aD aC aD aC
aE aE aE
0
Flowering Pod setting Pod filling
b Lima bean var. branca
180
Accumulated N
(mg N SDW-1)

135
aA bA bA
90
aB aC aB bC aB bC
45 bD aD bE bD bD bD aE
bE bF
0
Flowering Pod setting Pod filling
Rhizobium sp. ISOL-16 Bradyrhizobium sp. ISOL-19 Nitrogen control
Bradyrhizobium sp. ISOL-18 Bradyrhizobium sp. ISOL-35 Absolute control
Fig. 4 Accumulated nitrogen of ‘boca de moça’ (a) and ‘branca’ (b) lima different uppercase letters represent significant differences among
bean varieties inoculated with four different indigenous rhizobia, treatments in each variety whereas different lowercase letters indicate
Rhizobium sp. strain ISOL-16 or Bradyrhizobium sp. strain ISOL-18, significant differences between the two varieties (boca de moça and
ISOL-19 or ISOL-35, at flowering, pod setting or pod filling. Controls branca). SDW = shoot dry weight. All mean comparisons were
were uninoculated nitrogen-supplied plants (nitrogen control) and performed using Tukey’s test (P < 0.05)
uninoculated plants without nitrogen supply. In each harvest period,
Symbiotic performance, nitrogen flux and growth of lima bean
Accumulated nitrogen and the nitrogen content were posi-
tively modulated in lima bean varieties nodulated by
Bradyrhizobium in all harvest periods. Nitrogen flux between
rhizobia and host (often evaluated in terms accumulated nitro-
gen and the nitrogen content) reflect better BNF and also a
more efficient legume-rhizobia symbiosis (Figueiredo et al.
2016). Nitrogen fixed by rhizobia is transported to the host
legume, and the continuous flow of nitrogen during the veg-
etative and early reproductive stages is critical for plant
growth (Peix et al. 2015). It is important to note that among
the symbiotic pairs formed with Bradyrhizobium, lima bean
nodulated by Bradyrhizobium sp. ISOL-18 exhibited higher
accumulated nitrogen (Fig. 4) and nitrogen content
(Supplemental Fig. S3) compared to the other symbiotic pairs
independent of the lima bean variety utilized or the harvest
period.
Nitrogen is required for chlorophyll synthesis, which is cru-
cial for photosynthesis. Therefore, a decline in nitrogen avail-
ability reduces photosynthesis, thus reducing plant biomass and
reducing the supply of photosynthate to the nodules
(Kirschbaum 2011; Lobato et al. 2013). Lower values of nitro-
gen flow parameters (Fig. 4 and Supplemental Fig. S3) and
total chlorophyll (Supplemental Fig. S4) were observed in lima
bean nodulated by Rhizobium sp. ISOL-16 relative to the other
symbiotic pairs. Total chlorophyll, as determined by a chloro-
phyll meter, identified the plants with the greenest leaves, a
feature strongly correlated with photosynthetic efficiency
(Kirschbaum 2011). It is noteworthy that the plants nodulated
by Bradyrhizobium showed higher levels of total chlorophyll
than those nodulated by Rhizobium, so these plants likely main-
tain a higher photosynthetic rate for a longer period.
In this study, the highest nitrogen fixation efficiency values
were obtained during the pod-setting period, and the symbiot-
ic pairs formed with Bradyrhizobium were particularly effi-
cient (Fig. 5). The use of rhizobial strains that promote effec-
tive legume-rhizobia symbiosis with the aim of increasing soil
nitrogen is extremely important (Figueiredo et al. 2016). We
conclude that Bradyrhizobium is more efficient than
Rhizobium in establishing a successful symbiotic relationship
with lima bean varieties. Independent of lima bean variety, the
symbiotic pair formed by lima bean and Bradyrhizobium sp.
ISOL-18 stood out compared with the other tested symbiotic
pairs. These promising results highlight the potential for the
use of this strain of Bradyrhizobium as possible inoculants in
lima bean cultivation.
Acknowledgments The authors are grateful for the financial support of
the National Council of Technological and Scientific Development
(CNPq) and the Federal Agency for the Support and Evaluation of
Graduate Education (CAPES).
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