Symbiosis

DOI 10.1007/s13199-017-0473-8

Lima bean nodulates efficiently with Bradyrhizobium strains

isolated from diverse legume species
Elaine Martins da Costa 1 & Paula Rose de Almeida Ribeiro 1 & Wellington de Lima 1 &
Thiago Palhares Farias 1 & Fatima Maria de Souza Moreira 1

Received: 9 October 2016 / Accepted: 12 January 2017

# Springer Science+Business Media Dordrecht 2017

Abstract Lima bean (Phaseolus lunatus L.) is an important promote lima bean growth via biological nitrogen fixation in
legume species that establishes symbiosis with rhizobia, main- soil conditions.
ly of the Bradyrhizobium genus. The aim of this study was to
evaluate the efficiency of rhizobia of the genus Keywords Phaseolus lunatus L . Biological nitrogen
Bradyrhizobium in symbiosis with lima bean, in both fixation . Inoculant . Liming . Symbiosis
Leonard jars and in pots with a Latossolo Amarelo distrófico
(Oxisol). In the experiment in Leonard jars, 17 strains isolated
from nodules of the three legume subfamilies, Papilionoideae 1 Introduction
(Vigna unguiculata, Pterocarpus sp., Macroptilium
atropurpureum, Swartzia sp., and Glycine max), Lima bean (Phaseolus lunatus L.) is the second most econom-
Mimosoideae (Inga sp.), and Caesalpinioideae ically important crop among the four commercially exploited
(Campsiandra surinamensis) and two uninoculated controls, species of the Phaseolus genus worldwide (Fofana et al.
one with a low concentration (5.25 mg L−1) and another with a 1999). Although the use of lima bean is less than that of
high concentration (52.5 mg L−1) of mineral nitrogen (N) were common bean (P. vulgaris), it is one of the main legume spe-
evaluated. The six strains that exhibited the highest efficiency cies cultivated in tropical regions and represents an important
in Leonard jars, isolated from nodules of Vigna unguiculata alternative protein source for human consumption (Maquet
(UFLA 03–144, UFLA 03–84, and UFLA 03–150), et al. 1999). In Brazil, the Northeastern region accounts for
Campsiandra surinamensis (INPA 104A), Inga sp. (INPA approximately 95% of lima bean production, with a planted
54B), and Swartzia sp. (INPA 86A), were compared to two area of 41,318 ha, production of 17,078 tons of beans, and
uninoculated controls, one without and another with 300 mg grain yield of approximately 420 kg ha−1 for the year 2009
N dm−3 (NH4NO3) applied to pots with samples of an Oxisol (available at http://www.sidra.ibge.gov.br). This low grain
in the presence and absence of liming. In this experiment, yield may be attributed to the fact that most of the
liming did not affect nodulation and plant growth; the INPA production is from small-scale farmers with limited access to
54B and INPA 86A strains stood out in terms of shoot dry agricultural technology.
matter production and provided increases of approximately Like other species of the Phaseolus genus, lima bean is
48% in shoot N accumulation compared to the native rhizobia able to establish symbiosis with rhizobia (Thies et al. 1991;
populations. Our study is the first to indicate Bradyrhizobium Ormeno-Orrillo et al. 2006; Antunes et al. 2011; Matsubara
strains isolated from the three legume subfamilies are able to and Zúñiga-Dávila 2015). P. lunatus is a very diverse species
with many genotypes which vary in seed morphology, color,
and size. The few papers reporting N2 fixation in this species
* Fatima Maria de Souza Moreira
[email protected]
have usually restricted study to one cultivar. The symbiont
Bradyrhizobium genus has the main focus for research
1
Setor de Biologia, Microbiologia e Processos Biológicos do Solo,
(Ormeno-Orrillo et al. 2006; López-López et al. 2013;
Departamento de Ciência do Solo, Universidade Federal de Lavras, Matsubara and Zúñiga-Dávila 2015; Costa Neto et al. 2017).
Campus UFLA, Lavras, Minas Gerais 37200-000, Brazil However, information on the agronomic implications of this
 da Costa E.M. et al.

symbiosis is scarce, especially in Brazil, where only two stud- Lavras (Universidade Federal de Lavras - UFLA) were used.
ies evaluating the symbiotic efficiency of rhizobia strains in These strains were recently characterized by housekeeping
lima bean, have been performed under axenic conditions gene sequencing (Guimarães et al. 2015; Ribeiro et al.
(Antunes et al. 2011; Costa Neto et al. 2017). Thus, there is 2015), which indicated that they belong to different phyloge-
considerable need to expand studies, select, and recommend netic groups (Table 1). The origin and efficiency of these
strains to increase grain yield in this crop. strains in symbiosis with other legume species - cowpea
Among the rhizobia genera, Bradyrhizobium has attracted (Vigna unguiculata), siratro (Macroptilium atropurpureum),
attention in Brazil because it has broad geographic distribution or soybean (Glycine max) - is shown in Table 1. The ability
and establishes efficient symbiosis with many legume species of the rhizobia strains to fix nitrogen in symbiosis with differ-
of importance for agriculture, in pasture areas, and in forestry. ent legume species is a desirable characteristic since it facili-
Furthermore, natural and altered ecosystems within the tates commercialization by companies producing inoculants.
Amazon region accommodate a large diversity of
Bradyrhizobium strains (Moreira et al. 1993; Moreira et al. 2.2 Strain efficiency under axenic conditions
1998; Lima et al. 2009; Guimarães et al. 2012; Jaramillo
et al. 2013) that may be considered important sources of ge- The experiment was conducted at the SBMPBS (UFLA) from
netic resources for biotechnological application. April to May 2014. Mean values of maximum and minimum
In Brazil, rhizobia strains have been selected and autho- temperatures in the greenhouse were 38 and 14 °C, respectively,
rized by the Ministry of Agriculture (MAPA) for production during the experiment period. Treatments consisted of
of inoculants for 83 legume species, Among these, 62% have individual inoculations of 17 Bradyrhizobium strains and
inoculant strains from the genus Bradyrhizobium (details two uninoculated negative controls, one with a low
available at http://www.agricultura.gov.br). A main concentration (5.25 mg L−1) and the other with a high
advantage of this genus is that most of its nodulation and N2 concentration (52.5 mg L −1) of mineral nitrogen (N).
fixation genes are located on the chromosome, so that they The experiment followed a completely randomized
tend to be genetically stable. This characteristic is extremely design, with three replicates.
important for the selected strain not to lose its symbiotic A 1:2 mixture of sand (150 cm3) and vermiculite (300 cm3)
efficiency over the years. In addition to the symbiotic was placed at the top of Leonard jars. Hoagland nutrient solution
efficiency and genetic stability of a strain, the ability to (Hoagland and Arnon 1950), modified as described by
nodulate and fix nitrogen in symbiosis with different legume Guimarães et al. (2012), was placed at the bottom of the jars.
species and its competitiveness with native rhizobia The same N concentration used in the nutrient solution for the
populations should also be considered when selecting and control with low N concentration was used for the inoculated
recommending new rhizobia strains for a certain legume treatments. After preparation, the jars and nutrient solution were
species. Competitiveness of a strain with native rhizobia autoclaved for one hour at 1.5 kg cm−2 at 121 °C.
populations is one of the main factors determining the An accession of Bcriolo^ lima bean (white seed), one of the
response of a legume species to inoculation (Singleton and accessions often grown by farmers in Northeastern Brazil, was
Tavares 1986; Thies et al. 1991). Other factors, such as the used. Before sowing, the seeds were surface sterilized using 98%
genetic traits of both the macro- and microsymbiont, soil acid- ethyl alcohol (30 s) and 2% sodium hypochlorite (2 min).
ity, and nutrient availability, can also affect nodulation and the Seeds were successively washed in sterile distilled water,
BNF process (Hartwig 1998; Bonilla and Bolaños 2009; pre-germinated in sterile petri dishes containing filter
Moreira et al. 2010; Rufini et al. 2011). paper and wet cotton, and then kept for 48 h in a growth
The aim of this study was to evaluate the efficiency of chamber at 28 °C. Four seeds were sown in each Leonard
nitrogen-fixing bacteria of the Bradyrhizobium genus isolated jar and thinning was performed seven days after
from diverse legume species in symbiosis with lima bean un- emergence, leaving one plant per jar.
der axenic conditions and in soil. The bacterial strains were cultured in liquid culture medium 79
(Fred and Waksman 1928) under stirring at 110 rpm at 28 °C for
five days. In each inoculated treatment, 1 mL of inoculant at a
2 Materials and methods concentration of 1 × 108 bacterial cells mL−1 was added to each
seedling. After inoculation, a layer of paraffin sand (10 kg of sand,
2.1 Strains evaluated 1 L of chloroform, and 10 g of paraffin) was added to each pot to
avoid possible contamination. The nutrient solution was prepared,
In this study, 17 Bradyrhizobium strains from the collection of autoclaved, and reapplied to the pots periodically throughout the
the Sector of Biology, Microbiology, and Biological Processes experiment.
of the Soil (Setor de Biologia, Microbiologia e Processos At 45 days after sowing (onset of flowering) the SPAD (Soil
Biológicos do Solo - SBMPBS) of the Federal University of Plant Analysis Development) index, which represents an indirect
Lima bean nodulates efficiently with Bradyrhizobium strains

Table 1 Origin, symbiotic efficiency in other hosts and phylogenetic affiliation of the Bradyrhizobium strains used in this study

Strains Origin Symbiotic efficiency Phylogenetic Phylogenetic References

in axenic conditionsa groups c,d Affiliation
Region/State Land use Host plant and/or in soilb
systems

UFLA 03–84 Amazonian /RO Pasture Vigna V. unguiculata (AI) a, b Ic Bradyrhizobium sp. Soares et al. (2006)
unguiculata
UFLA 03–144 Amazonian/AM Agriculture V. unguiculata V. unguiculata (IE) a Ic Bradyrhizobium sp. Guimarães et al. (2012)
UFLA 03–268 Amazonian/AM Agroforestry V. unguiculata V. unguiculata (IE) a Ic Bradyrhizobium sp. Jaramillo et al. (2013)
INPA 237B Amazonian/AM Forestry Pterocarpus sp. M. atropurpureum (E)a Ic Bradyrhizobium sp. Guimarães et al. (2015)
INPA 104A Amazonian/AM Forestry Campsiandra M. atropurpureum (E) a Ic Bradyrhizobium sp. Guimarães et al. (2015)
surinamensis
UFLA 03–290 Amazonian/AM Agroforestry V. unguiculata V. unguiculata (I) a IV c Bradyrhizobium sp. Jaramillo et al. (2013)
UFLA Amazonian/AM Agriculture Macroptilium M. atropurpureum (E) IV c
a
Bradyrhizobium sp. Florentino et al. (2009)
04–0212 atropurpureum
INPA 54B Amazonian/AM Forestry Inga sp. M. atropurpureum (E) a Vc Bradyrhizobium sp. Moreira et al. (1998)
INPA 86A Amazonian/AM Forestry Swartzia sp. M. atropurpureum (E) a Vc Bradyrhizobium sp. Guimarães et al. (2015)
UFLA 03–150 Amazonian/AM Agriculture V. unguiculata V. unguiculata (E) a II c Bradyrhizobium sp. Guimarães et al. (2012)
UFLA 03–197 Amazonian/AM Agriculture V. unguiculata V. unguiculata (E) a II c Bradyrhizobium sp. Guimarães et al. 2012)
UFLA 03–153 MG Bauxite Mining V. unguiculata V. unguiculata (E) a, b Ic Bradyrhizobium sp. Soares et al. (2014)
UFLA 03–164 MG Bauxite Mining V. unguiculata V. unguiculata (E) a, b Ic Bradyrhizobium sp. Soares et al. (2014)
UFLA 03–320 MG Agriculture V. unguiculata V. unguiculata (E) a IV c Bradyrhizobium sp. Rufini et al. (2013)
UFLA 03–321 MG Agriculture V. unguiculata V. unguiculata (E) a IV c Bradyrhizobium sp. Rufini et al. (2013)
UFLA 06–24 Cerrado/PI Agriculture Glycine max G. max (E) a Single d Bradyrhizobium sp. Ribeiro et al. (2015)
UFLA 06–13 Cerrado/PI Agriculture G. max G. max (E) a G-II d Bradyrhizobium sp. Ribeiro et al. (2015)

RO Rondôna, AM Amazonas, MG Minas Gerais, PI Piauí

AI Approved as inoculant by the Brazilian Ministry of Agriculture, Livestock and Supply (Ministério da Agricultura Pecuária e Abastecimento)
IE intermediate efficiency (Shoot dry matter of the treatment inoculated with the tested strain < to that of the uninoculated control with nitrogen
supplementation, and > to that of the uninoculated control with low nitrogen concentration)
I inefficient (Shoot dry matter of the treatment inoculated with the tested strain = to that of the uninoculated control with low nitrogen concentration)
E efficient = (Shoot dry matter of the treatment inoculated with the tested strain = to that of the uninoculated control with nitrogen supplementation)
a
Jars with nutrient solution, with or without sand and vermiculate, esterilized in autoclave
b
Non-sterile soil
c
Grouping according to the phylogenetic analysis of the housekeeping genes by Guimarães et al. (2015)
d
Grouping according to the phylogenetic analysis of the housekeeping genes by Ribeiro et al. (2015)

measurement of leaf chlorophyll content, was determined. A total 2.3 Strain efficiency in pots with soil
of 15 readings were taken from the last fully developed trifoliate
leaf for each plant using a Minolta SPAD-502 chlorophyll meter. The experiment was conducted from October to November
After the readings, the plants were collected to evaluate the 2014 in a greenhouse at the SBMPBS (UFLA). Soil
following variables: number of nodules (NN), nodule dry matter characterized as a Latossolo Amarelo distrófico (Oxisol)
(NDM), shoot dry matter (SDM), root dry matter (RDM), total collected in the municipality of São Luís, MA, in a site at
dry matter (TDM), and shoot nitrogen accumulation (SNA). The the Federal Institute of Education, Science, and Technology
nodules, shoots, and roots were placed in paper bags and dried in of Maranhão (Instituto Federal de Educação, Ciência e
a forced air oven at 60 °C to constant weight to determine the Tecnologia do Maranhão) (altitude 27 m, 2°36′37″S and
NDM, SDM, and RDM. Shoot N content was determined using 4°16′18″W) was used. The state of Maranhão is one of the
the semi-micro Kjedahl method. The SNA was calculated by main lima bean producers in Northeastern Brazil.
multiplying the SDM (mg) by the N content (%) / 100. Additionally, this soil class is predominant within the state
According to the Shapiro-Wilk test, the experimental data and throughout Brazil. The site where the samples were
exhibited normal distribution and were subjected to analysis of collected has a history of annual corn (Zea mays L.)
variance using the SISVAR 5.3 statistical analysis program cultivation under a conventional tillage system. Every three
(Ferreira 2011). Mean values were grouped by the Scott-Knott years, liming and N-P-K fertilization (100–80-90) is
test at 5% probability. The NN and NDM data were transformed performed and there is no history of application of any type
into the square root of (Y + 0.5). Pearson correlation coefficients of inoculant. Liming was last performed at this site two
were estimated at 1% and 5% probability levels. years before this study.
 da Costa E.M. et al.

Soil samples were collected at a depth of 0–20 cm and then 3 Results

crushed, homogenized, passed through a 4-mm sieve, and
placed in pots (3.5 dm3 capacity). Before implementing the 3.1 Strain efficiency under axenic conditions
experiment, the soil had the following characteristics: pH in
H2O, 6.0; P (Mehlich 1), 2.3 mg dm−3; K+, 20 mg dm−3; Ca2+ , The treatments had effects (p < 0.05) on all the variables evaluated
1.60 cmolc dm−3; Mg2+, 0.60 cmolc dm−3; Al3+, 0.00 cmolc (Table 2). There was no nodulation in the uninoculated controls,
dm−3; H + Al, 2.32 cmolc dm−3; sum of exchangeable bases, indicating that there was no contamination in the experiments.
2.25 cmolc dm−3; cation exchange capacity, 2.25 cmolc dm−3; Only four strains did not nodulate lima bean (UFLA 03–268,
cation exchange capacity at pH 7.0, 4.57 cmolc dm−3; alumi- UFLA 03–290, UFLA 06–24, and UFLA 06–13). Of the strains
num saturation, 0.00%; base saturation, 49.26%; organic mat- that nodulated, the mean NN values per plant ranged from 15 to
ter, 1.87 dag kg−1; clay, 13 dag kg−1; silt, 5 dag kg−1; and sand, 506 for the treatments inoculated with the UFLA 03–153 and
82 dag kg−1. UFLA 03–144 strains, respectively. The UFLA 03–84, UFLA
The experiment followed a randomized block design 03–144, INPA 104A, INPA 54B, UFLA 03–150, UFLA 03–
with four replicates in an 8 × 2 factorial arrangement 197, and UFLA 03–164 strains promoted higher NN (p < 0.05)
[8 N sources and 2 liming levels (with and without than the other strains. The highest (p < 0.05) NDM values were
liming)]. The following N sources were used: individual obtained in the treatments inoculated with the UFLA 03–144 and
inoculation of the 6 Bradyrhizobium strains that exhibited INPA 104A strains. The UFLA 03–84, INPA 54B, INPA 86A,
highest efficiency in the experiment in Leonard jars UFLA 03–150, and UFLA 03–197 strains formed a second group
(UFLA 03–84, UFLA 03–144, INPA 104A, INPA 54B, that also stood out (p < 0.05) in terms of NDM production.
INPA 86A, and UFLA 03–150) and 2 uninoculated Of the 17 strains evaluated, the highest (p < 0.05) SDM, TDM,
controls, one without and another with mineral N fertilizer SPAD index, and SNA values were obtained from the seven
(300 mg dm−3). The lime application rate in the liming strains that also stood out in terms of NDM production (UFLA
treatments was calculated according to the base saturation 03–84, UFLA 03–144, INPA 104A, INPA 54B, INPA 86A,
method to raise the saturation to 60% using calcium UFLA 03–150, and UFLA 03–197) (Table 2). Positive correla-
carbonate (CaCO3) and magnesium carbonate (MgCO3) tions (p < 0.01) were detected between NDM and the other var-
at a ratio of 4:1, respectively. The soil was kept moist iables, except for RDM. There was also high correlation
and incubated for 30 days before planting. In all plots, (p < 0.01) between the SPAD index and SNA (Table 3).
the following fertilization was carried out: 300, 300, 40, The INPA 54B strain promoted higher (p < 0.05) SDM (7.29 g
5.0, 1.5, 3.6, 0.8, and 0.15 mg dm−3 of K, P, S, Zn, Cu, plant−1), TDM (8.77 g plant−1), and SNA (0.26 g plant−1) values
Mn, B, and Mo, respectively. For the control with mineral than the other strains and the control with high N concentration
N, NH4NO3 (300 mg N dm−3) was provided, divided into (Table 2). The UFLA 03–144 strain was the second most efficient
three applications. for these variables, promoting higher (p < 0.05) values than the
The lima bean seeds and the method for disinfecting control with high N concentration and than the other strains. The
them were the same as used in the previous experiment. treatments inoculated with the UFLA 03–84, INPA 104A, and
Six seeds were sown per pot, and each inoculated INPA 86A strains formed a third group with higher (p < 0.05)
treatment received 1 mL of the inoculant at 1 × 10 8 SDM, TDM, and SNA values than the control with high N con-
bacterial cells mL −1 on each seed. Eight days after centration. The treatments inoculated with the UFLA 03–150 and
emergence, the plants were thinned to two seedlings per UFLA 03–197 strains exhibited similar and lower (p < 0.05)
pot. values of these variables, respectively, compared to the control
The plants were collected at 45 days after sowing (during with high N concentration. The other strains were inefficient in
flowering) to evaluate the following variables: number of BNF, achieving SDM and SNA values similar (p < 0.05) to the
nodules (NN), nodule dry matter (NDM), shoot dry matter control with low N concentration.
(SDM), root dry matter (RDM), total dry matter (TDM), and For the RDM, 82% of the strains evaluated exhibited
shoot nitrogen accumulation (SNA). These variables were higher (p < 0.05) values than the control with low N concen-
obtained by the procedure described in the previous tration (Table 2). The INPA 54B strain stood out (p < 0.05)
experiment. According to the Shapiro-Wilk test, the compared to the other strains and led to a response similar
experimental data exhibited normal distribution and were (p < 0.05) to the control with high N concentration. The
subjected to analysis of variance using the SISVAR 5.3 UFLA 03–144, INPA 237B, INPA 104A, and UFLA
statistical analysis program (Ferreira 2011). Mean values 03–153 nodulating strains, together with three strains that
were grouped by the Scott-Knott test at 5% probability. did not nodulate lima bean (UFLA 03–290, UFLA 06–24,
The NN and NDM data were transformed into the square and UFLA 06–13), formed a second group with higher
root of (Y + 0.5). Pearson correlation coefficients were (p < 0.05) RDM values than the other strains and the
estimated at 1% and 5% probability levels. control with low N concentration, though these values
Lima bean nodulates efficiently with Bradyrhizobium strains

Table 2 Number of nodules (NN), nodule dry matter (NDM), shoot dry matter (SDM), root dry matter (RDM), total dry matter (TDM), SPAD index
(SPAD) and shoot nitrogen accumulation (SNA) obtained in lima bean plants in Leonard jars with different nitrogen sources at 45 days after sowing

N sources NN NDM SDM RDM TDM SPAD SNA

- ——————————— g plant−1 ————————————— - g plant−1

Without I + High N (52.5 mg L−1) a 0d 0e 4.45 d 1.60 a 6.04 c 27.23 c 0.09 f

Without I + Low N (5.25 mg L−1) a 0d 0e 1.49 f 0.83 d 2.27 g 14.06 e 0.02 g
UFLA 03–84 + Low N 352 a 0.34 b 5.03 c 1.06 c 6.09 c 30.53 b 0.20 c
UFL 03–144 + Low N 506 a 0.57 a 6.57 b 1.24 b 7.82 b 32.43 b 0.23 b
UFLA 03–268 + Low N 0d 0e 1.65 f 0.98 c 2.63 g 17.30 d 0.02 g
INPA 237B + Low N 51 c 0.08 e 1.59 f 1.14 b 2.73 g 15.16 e 0.02 g
INPA 104A + Low N 464 a 0.51 a 5.35 c 1.21 b 6.57 c 27.53 c 0.19 c
UFLA 03–290 + Low N 0d 0e 1.59 f 1.17 b 2.75 g 17.60 d 0.02 g
UFLA 04–2012 + Low N 207 b 0.12 d 1.91 f 0.79 d 2.70 g 18.46 d 0.02 g
INPA 54B + Low N 435 a 0.41 b 7.29 a 1.48 a 8.77 a 37.73 a 0.26 a
INPA 86A + Low N 220 b 0.33 b 5.24 c 1.07 c 6.31 c 33.26 b 0.20 c
UFLA 03–150 + Low N 471 a 0.36 b 4.04 d 1.08 c 5.12 d 31.03 b 0.15 d
UFLA 03–197 + Low N 336 a 0.35 b 3.48 e 0.97 c 4.45 e 31.26 b 0.11 e
UFLA 03–153 + Low N 15 c 0.01 e 2.34 f 1.15 b 3.50 f 14.00 e 0.03 g
UFLA 03–164 + Low N 362 a 0.21 c 1.83 f 0.82 d 2.65 g 13.16 e 0.02 g
UFLA 03–320 + Low N 283 b 0.05 e 1.92 f 1.01 c 2.93 g 15.20 e 0.02 g
UFLA 03–321 + Low N 274 b 0.12 d 1.93 f 0.91 d 2.84 g 19.13 d 0.03 g
UFLA 06–24 + Low N 0d 0e 2.01 f 1.34 b 3.35 f 17.26 d 0.03 g
UFLA 06–13 + Low N 0d 0e 1.80 f 1.23 b 3.04 g 17.73 d 0.02 g
CV (%) 18.66 3.40 9.35 12.09 8.27 11.20 10.33
a
I= inoculation, Nitrogen sources used in the nutrient solution: Ca(NO3)2·4H2O, KNO3 NH4H2PO4
In each column, means followed by the same letter are not statistically different and belong to the same group, according to the Scott–Knott test at
P < 0.05

were lower (p < 0.05) than the value of the control with The highest (p < 0.05) SNA value was obtained in the
high N concentration. uninoculated control with mineral N (Table 4). The INPA
54B and INPA 86A strains were most efficient in N2 fixation,
3.2 Strain efficiency in pots with soil promoting higher (p < 0.05) SNA than the other strains and
the uninoculated control without mineral N. The UFLA 03–84
There were effects (p < 0.05) only of nitrogen sources on the and UFLA 03–144 strains also promoted higher (p < 0.05)
NN, NDM, SDM, TDM, and SNA variables (Table 4). The SNA than the uninoculated control without mineral N. The
highest (p < 0.05) NN values were observed in the treatments treatments inoculated with the INPA 104A and UFLA 03–150
inoculated with the UFLA 03–144 and INPA 86A strains; strains exhibited similar and lower (p < 0.05) SNA,
however, for NDM, there was no difference between the in- respectively, than the uninoculated control without N.
oculated treatments and the uninoculated control without min- Positive correlations (p < 0.05) were observed between the
eral N. The plants fertilized with mineral N did not nodulate. NN, NDM, and SNA (Table 3).
All strains, except UFLA 03–150, promoted higher SDM Regarding RDM, there was an interaction (p < 0.05) between
and TDM production (p < 0.05) than the uninoculated control the liming and the N sources (Table 5). The UFLA 03–144 and
without mineral N (Table 4). However, no strain was similar UFLA 03–150 strains induced higher (p < 0.05) RDM
or superior (p < 0.05) to the uninoculated control with mineral production in the presence of liming, whereas the INPA 104A
N. The INPA 54B and INPA 86A strains stood out (p < 0.05) strain promoted higher (p < 0.05) RDM production in the
from the others in SDM production and promoted 34 and 36% absence of liming. Among the N sources, in the absence of
increases, respectively, compared with the production liming, the treatments inoculated with the UFLA 03–84 and
obtained from the uninoculated control without mineral N. INPA 104A strains exhibited higher (p < 0.05) RDM production
The treatments inoculated with the UFLA 03–84, UFLA than the other inoculated treatments and the controls with and
03–144, and INPA 104A strains promoted similar (p < 0.05) without mineral N application. In the presence of liming, the
SDM production. inoculated treatments, except for the one inoculated with the
 da Costa E.M. et al.

Table 3 Pearson’s correlation coefficients between the parameters The six strains that exhibited the highest efficiency in N2 fixation
studied in the experiments in Leonard jars and in pots with non
in symbiosis with lima bean in Leonard jars were isolated from
sterilized soil (Oxisol)
nodules of Vigna unguiculata (UFLA 03–84, UFLA 03–144, and
NDM SDM RDM TDM SNA SPAD UFLA 03–150), Campsiandra surinamensis (INPA 104A), Inga
sp. (INPA 54B), and Swartzia sp. (INPA 86A) in Amazon soils.
Leonard jars (axenic conditions)
These strains were also efficient in symbiosis with cowpea
NN 0.85** 0.59** -0.091ns 0.55** 0.66** 0.58**
**
(UFLA 03–144, UFLA 03–84, and UFLA 03–150) and siratro
NDM 0.80 0.062ns 0.76** 0.85** 0.74**
(INPA 104A, INPA 54B, and INPA 86A) in previous studies
SDM 0.46* 0.99** 0.96** 0.88**
(Table 1), indicating the potential of Amazon soils as a source
RDM 0.55** 0.35* 0.37*
of genetic resources for biotechnological applications. Although,
TDM 0.95** 0.87**
seed N may have contributed to plant N, this contribution was
SNA 0.91** similar in all treatments. Thus, the differences observed can be
Pots with non sterilized soil (Oxisol) considered as being due to the treatments, i.e., the strains
NN 0.76** 0.15ns -0.09ns 0.13ns 0.32* - inoculated.
ns ns ns
NDM 0.23 0.06 0.25 0.36* - The UFLA 03–84 strain is currently authorized by the MAPA
SDM 0.17 ns 0.95ns 0.84** - as a cowpea inoculant (information available at http://www.
RDM 0.43** 0.11 ns - agricultura.gov.br) and its symbiotic efficiency was also
TDM 0.80** - observed in pigeon pea (Cajanus cajan L.) in Leonard jars
SNA - (Rufini et al. 2014). In contrast, the UFLA 03–153, UFLA 03–
** 164, UFLA 03–320, and UFLA 03–321 strains, which are
p < 0.01; * p < 0.05; ns not significant
efficient in symbiosis with cowpea (Rufini et al. 2013; Soares
et al. 2014), and the INPA 237B and UFLA 04–0212 strains,
INPA 104A strain, promoted higher (p < 0.05) RDM production
which form efficient symbiosis with siratro (Table 1), were
than the controls with and without mineral N application.
inefficient in symbiosis with lima bean in this study, indicating
that BNF is affected by the intrinsic characteristics of the
symbionts, as reported by Hartwig (1998).
4 Discussion High symbiotic efficiency of isolates (genetically unidentified)
of lima bean nodules inoculated in this crop was reported by
Selecting rhizobia strains efficient in biological nitrogen fixation Antunes et al. (2011) in an experiment conducted in Leonard jars.
(BNF) in symbiosis with lima bean is an important strategy to These authors also detected positive correlations (p < 0.05)
economically and sustainably increase the grain yield of this crop. between NDM and SDM production and SNA, corroborating

Table 4 Number of nodules

(NN), nodule dry matter (NDM), Factors NN NDM SDM TDM SNA
shoot dry matter (SDM), root dry N° pot−1 —————————— g pot−1 ——————————
matter (RDM), total dry matter
(TDM) and shoot nitrogen N Sources
accumulation (SNA) obtained in UFLA 03–84 169 b 0.29 a 3.93 c 5.47 b 0.12 c
lima bean plants in pots with UFLA 03–144 250 a 0.34 a 3.94 c 5.31 b 0.13 c
samples of a Oxisol according to
the nitrogen sources and with or INPA 104A 178 b 0.29 a 3.97 c 5.86 b 0.10 d
without liming at 45 days after INPA 54B 202 b 0.34 a 4.47 b 5.64 b 0.15 b
sowing INPA 86A 309 a 0.34 a 4.53 b 5.77 b 0.15 b
UFLA 03–150 184 b 0.26 a 3.27 d 4.51 c 0.06 e
Without N without I 218 b 0.30 a 3.34 d 4.47 c 0.10 d
With N (300 mg dm−3) 0c 0b 6.69 a 7.91 a 0.35 a
Liming
Without liming 187 a 0.27 a 4.23 a 5.52 a 0.15 a
With liming 190 a 0.27 a 4.29 a 5.63 a 0.14 a
CV (%) 23.15 5.27 12.23 9.54 16.52

N Nitrogen, I Inoculation
Means followed by the same letters within the columns are not significantly different from each other by the Scott-
Knott test at p < 0.05
Lima bean nodulates efficiently with Bradyrhizobium strains

Table 5 Root dry matter (RDM) of lima bean plants in pots with a common bean and found positive responses (p < 0.05) to
Oxisol according to different nitrogen sources with or without liming
liming on nodulation, plant growth, and N 2 fixation.
N sources RDM (g pot−1) However, these results were observed in a Oxisol with an
initial pH (H 2 O) of 5.1 and a base saturation of 21%.
Without liming With liming Liming is an important practice for two reasons: it raises soil
UFLA 03–84 1.54 aA 1.55 aA
pH, reducing acidity, and increases base saturation, supplying
calcium and magnesium, which are important nutrients for
UFLA 03–144 1.17 bB 1.51 aA
plant and diazotrophic bacterial development and,
INPA 104A 1.48 aA 1.13 bB
consequently, for establishment of the symbiosis (Norris
INPA 54B 1.29 bA 1.49 aA
1958; Lodeiro et al. 1995). In the present study, the relatively
INPA 86A 1.14 bA 1.37 aA
high pH (6.0) and Ca+2 (1.60 cmolc dm−3) and Mg2+ (0.60
UFLA 03–150 1.12 bB 1.37 aA
cmolc dm−3) levels present in the soil probably adequately
Without N without I 1.13 bA 1.14 bA
sustained the plants during the experimental period and could
With N (300 mg dm−3) 1.27 bA 1.15 bA
explain why the plant did not respond to the addition of lime.
Means 1.27 A 1.34 A
However, two strains (UFLA 03–144 and UFLA 03–150)
CV (%) 12.90
increased (p < 0.05) the RDM in response to liming.
N nitrogen, I inoculation The lack of correlation (p > 0.05) between NDM and SDM
Means followed by the same letters, lowercase letters in columns and and the lower correlation (p < 0.05) between NDM and SNA
uppercase in the lines are not significantly different from each other by in the experiment in pots with soil differs from the results
the Scott-Knott test at p < 0.05 obtained in Leonard jars. This may be due mainly to the
effects of edaphic factors. The absence of nodulation in the
the results obtained in the present study. Similar correlation results uninoculated control with mineral N in pots with soil indicates
were reported by Ferreira et al. (2012) when working with a mitigating role of nitrogen in lima bean nodulation, which
common bean (Phaseolus vulgaris L.) inoculated with rhizobia has also been observed in other legume species, such as
strains. cowpea (Costa et al. 2014), common bean (Rufini et al.
The SPAD index, which represents an indirect measure 2011), and pigeon pea (Rufini et al. 2014). The similarity in
of leaf chlorophyll content, has been positively correlated NDM between the uninoculated control without mineral N
with shoot N accumulation, grain yield, and/or shoot dry and the inoculated treatments is evidence for the effective
matter in some legume species, such as common bean, nodulating capacity of the native rhizobia populations present
soybean, and cowpea (Fritschi and Ray 2007; Remans in the soil under study. Despite this similarity, four strains
et al. 2008; Jaramillo et al. 2013). However, for lima bean, (UFLA 03–144, UFLA 03–84, INPA 54B, and INPA 86A)
our study is the first to report this index and its excellent exhibited a competitive ability and promoted increased
correlation with both SNA and SDM production. Thus, we (p < 0.05) SDM and TDM production and SNA. This
suggest evaluating the SPAD index during the process of confirms the potential of some strains for use as lima bean
selecting rhizobia strains for this crop, especially in the first inoculants, especially INPA 54B and INPA 86A, which were
steps because when large numbers of strains are tested, the the most efficient in pot trials.
typical procedure, the cost for N analyses becomes very The rapid plant response to mineral N shows that no strain
expensive. is equal to the control fertilized with high NH 4 NO 3
The INPA 237B, UFLA 03–153, and UFLA 03–320 concentration (300 mg N dm−3), as the establishment and
strains that were inefficient in BNF and the four strains that functioning of a symbiosis takes time. However, once the
did not nodulate lima bean (UFLA 03–268, UFLA 03–290, symbiosis is established, the performance of the inoculated
UFLA 06–24, and UFLA 06–13) in Leonard jars most likely treatments can equal that of the mineral N treatment. This
increased (p < 0.05) the RDM production by acting in stage is reached during the grain production phase in crops
biological processes other than BNF. Although the main such as cowpea (Soares et al. 2006; Ferreira et al. 2013). There
function of the Bradyrhizobium genus has already been is no data yet regarding this in lima bean.
reported as BNF, this genus is quite versatile and may also Our study is the first to report the symbiotic efficiency of
act in other plant growth-promoting processes, such as rhizobia strains with lima bean in an experiment conducted in
inorganic phosphate solubilization and phytohormone soil. It constitutes the first step in selecting strains to
production (Boiero et al. 2007; Marra et al. 2011; recommend strains for lima bean inoculation. In future
Oliveira-Longatti et al. 2014). experiments we plan to evaluate INPA 54B and INPA 86A
In the experiment in pots with soil, the absence of a liming as lima bean inoculants under field conditions taking into
effect on lima bean nodulation and shoot growth differs from account other edaphoclimatic conditions to confirm the
the results obtained by Rufini et al. (2011). They worked with symbiotic efficiency of these strains.
 da Costa E.M. et al.

5 Conclusions Florentino LA, Guimarães AP, Rufini M, Silva K, Moreira FMS (2009)
Sesbania virgata Stimulates the occurrence of its microsymbiont in
soils but does not inhibit microsymbionts of other species. Sci Agric
Strains isolated from nodules of hosts - Vigna unguiculata 66:667–676. doi:10.1590/S0103-90162009000500012
(UFLA 03–144 and UFLA 03–84), Campsiandra Fofana B, Baudoin JP, Vekemans X, Debouck DG, Du Jardin P (1999)
surinamensis (INPA 104A), Inga sp. (INPA 54B), and Molecular evidence for an Andean origin and a secondary gene pool
Swartzia sp. (INPA 86A) - belonging to the three legume for the lima bean (Phaseolus lunatus L.) using chloroplast DNA.
Theor Appl Gen 98:202–212. doi:10.1007/s001220051059
subfamilies are efficient in symbiosis with lima bean under Fred EB, Waksman SA (1928) Laboratory manual of general microbiol-
axenic conditions and in pots with soil. Liming does not affect ogy: with special reference to the microorganisms of the soil.
lima bean nodulation and shoot growth in the soil evaluated McGraw-Hill, New York
during the experimental period. The INPA 54B and INPA 86A Fritschi FB, Ray JD (2007) Soybean leaf nitrogen, chlorophyll content,
and chlorophyll a/b ratio. Photosynthetica 45:92–98. doi:10.1007
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accumulation compared to the other strains and the native Moreira FMS (2012) Genetic and symbiotic diversity of nitrogen-
rhizobial populations, exhibiting potential for use as lima bean fixing bacteria isolated from agricultural soils in the western
inoculants. Amazon by using cowpea as the trap plant. Appl Environ Microb
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Guimarães AA, Florentino LA, Almeida KA, Lebbe L, Silva KB,
Acknowledgements We thank the Coordination for the Improvement Wi l l e m s A , M o r e i r a F M S ( 2 0 1 5 ) H i g h d i v e r s i t y o f
of Higher Education Personnel [Coordenação de Aperfeiçoamento de Bradyrhizobium strains isolated from several legume species and
Pessoal de Nível Superior (CAPES)] and the National Council for land uses in Brazilian tropical ecosystems. Syst Appl Microbiol
Scientific and Technological Development [Conselho Nacional de 38:433–441. doi:10.1016/j.syapm.2015.06.006
Desenvolvimento Científico e Tecnológico (CNPq)] for financial support Hartwig UA (1998) The regulation of symbiotic N2 fixation: a conceptual
and for granting scholarships. We also thank the Federal Institute of model of N feedback from the ecosystem to the gene expression
Education, Science and Technology of Maranhão [Instituto Federal de level. Perspect Plant Ecol 1:92–120. doi:10.1078/1433-8319-00054
Educação, Ciência e Tecnologia do Maranhão (IFMA)] for granting soil Hoagland DR, Arnon DI (1950) The water culture method for growing
for the experiment. plants without soil. Agric Exp Stn (Circular, n.347). Univ. of
California, Berkeley, California
Jaramillo PMD, Guimarães AA, Florentino LA, Silva KB, Nóbrega RSA,
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