Microbiological Research 239 (2020) 126522

Contents lists available at ScienceDirect

Microbiological Research
journal homepage: www.elsevier.com/locate/micres

Plant growth-promoting bacteria isolated from wild legume nodules and T

nodules of Phaseolus vulgaris L. trap plants in central and southern Mexico
Erika Yanet Tapia-García, Verónica Hernández-Trejo, Joseph Guevara-Luna,
Fernando Uriel Rojas-Rojas, Ivan Arroyo-Herrera, Georgina Meza-Radilla,
María Soledad Vásquez-Murrieta, Paulina Estrada-de los Santos*
Instituto Politécnico Nacional, Escuela Nacional de Ciencias Biológicas, Prol. Carpio y Plan de Ayala s/n, Col. Santo Tomás, C.P. 11340, Mexico City, Mexico

A R T I C LE I N FO A B S T R A C T

Keywords: Central southern Mexico contains highly diverse legumes. In this study, nodule-associated bacteria (NAB) were
Nodule associated bacteria isolated from wild legume nodules and from nodules on Phaseolus vulgaris plants used as a plant-trap in soils from
Nodulation the same areas as the wild legumes. The bacteria were identified through the 16S rRNA gene sequence analysis,
Biological nitrogen fixation tested for plant growth-promoting (PGP) activities and the production of antimicrobial compounds, and ana-
Mimosa
lyzed for potential nodulation by amplifying the nodC gene. Several genera with PGP activity were isolated from
Rhizosphere
legume nodules, including Achromobacter, Acinetobacter, Bacillus, Brevibacillus, Brevibacterium, Burkholderia,
Cupriavidus, Dyella, Ensifer, Enterobacter, Herbaspirillum, Kosakonia, Labrys, Microbacterium, Moraxella,
Paraburkholderia, Pseudomonas, Rhizobium, Stenotrophomonas; and Aeromonas, Marinococcus Pseudarthrobacter
and Pseudoxanthomonas were found in plant legume nodules for the first time. Pseudomonas was the most
common bacteria, and Mimosa pudica was colonized by the largest number of genera (6 different genera). A
Burkholderia strain from the Burkholderia cepacia complex and a firmicutes strain harbor the nodC gene, iden-
tifying them as potential novel nodulating bacteria and showing that most of the strains isolated in this study
were NAB. The most frequent PGP activity identified among the strains isolated from wild legumes was IAA
synthesis. Two bacteria, Stenotrophomonas sp. and Rhizobium sp., synthesized more than 250 μg/ml, which is
more than the level of synthesis reported in this study for Azospirillum brasilense Sp7 (59.77 μg/ml). Nitrogen
fixation and antimicrobial compound production were not common, but the production of siderophores was
frequently found among all the strains. This study shows that diverse NAB with PGP activity are very common in
the legume nodules from central southern Mexico.

1. Introduction legumes remain mostly unexplored (Selvakumar et al., 2008; Sánchez-

Some endophytes are plant growth-promoting (PGP) bacteria that
are important in plant development in challenging environmental
conditions (Muresu et al., 2019). These microorganisms can coexist
with Alpha- or Beta-rhizobia in the legume nodules (Ibáñez et al., 2017;
Muresu et al., 2008) but are themselves unable to nodulate. Some of
them can fix nitrogen (BNF, biological nitrogen fixation) or carry out
other PGP activities, and are therefore called nodule-associated bacteria
(NAB) or non-rhizobial bacteria (Ibáñez et al., 2017; Clua et al., 2018;
Martínez-Hidalgo and Hirsch, 2017).
Wild legumes can associate with noncultivable rhizobia and some
cultivable endophytes; in contrary, agricultural legumes contain several
cultivable rhizobia but fewer endophytes (Muresu et al., 2019). Gen-
erally, the study of NAB is derived from agricultural legumes, but wild
Cruz et al., 2019). In any case, the role of NAB within the nodule has
been understudied, and whether they are important to plant develop-
ment is unknown (Martínez-Hidalgo and Hirsch, 2017). Understanding
the strategies used by legumes to select the best partners for improving
BNF is important for sustainable agriculture (Clua et al., 2018). This
knowledge would be useful in formulating biofertilizers based on single
strains or combining NAB and rhizobia, thereby diminishing the use of
chemical fertilizers (Martínez-Hidalgo and Hirsch, 2017).
In Mexico the study of Alpha-rhizobia, in Phaseolus vulgaris L., has
been extensive, and several bacterial species have been described
within the genera Rhizobium, such as Rhizobium acidisoli, Rhizobium
endophyticum, Rhizobium esperanzae, Rhizobium etli, Rhizobium hi-
dalgoense, Rhizobium mesoamericanum and Rhizobium tropici (Martínez-
Romero et al., 1991; Segovia et al., 1993; López-López et al., 2010,

⁎
Corresponding author.
E-mail address: pestradadelossantos@gmail.com (P. Estrada-de los Santos).

https://doi.org/10.1016/j.micres.2020.126522
Received 28 February 2020; Received in revised form 24 May 2020; Accepted 30 May 2020
Available online 13 June 2020
0944-5013/ © 2020 Elsevier GmbH. All rights reserved.
E.Y. Tapia-García, et al.
2012; Cordeiro et al., 2017; Yan et al., 2017; Román-Ponce et al.,
2016). R. etli was found in nodules of Acacia farnesiana and Acacia
cochliacantha used as trap plants with soil from Morelos state in Mexico
(Miranda-Sánchez et al., 2016). Acaciella angustissima collected from
the Sumidero Canyon National Park in Chiapas, Mexico was found
nodulated by Sinorhizobium chiapanecum, Ensifer mexicanum, Rhizobium
tropici, Mesorhizobium plurifarium and Agrobacterium tumefaciens (now
Agrobacterium radiobacter) (Rincón-Rosales et al., 2009). In Desmodium
nodules Bradyrhizobium was found in Northern of Mexico (Parker,
2002). In Mimosa, R. etli, R. mesoamericanum, Burkholderia sp., Ensifer
sp., and Rhizobium sp. have been isolated in Mexico (Wang et al., 1999;
López-López et al., 2012; Bontemps et al., 2016). Also, in Mexico Rhi-
zobium grahamii was isolated from nodule of Leucaena leucocephala
(López-López et al., 2012). However, P. vulgaris L. is the second most
important crop in the world, after maize (Avelar et al., 2012; Cordeiro
et al., 2017). Previous works showed that P. vulgaris L. is a promiscuous
legume that can be nodulated by different rhizobia (Martínez-Romero,
2003), however, none these studies focused on the isolation of NAB.
In a recent study the NAB Enterobacter sp. NOD1 isolated from
Mimosa pudica L. nodules in Chiapas (Mexico) was characterized
(Sánchez-Cruz et al., 2019). This strain was unable to form nodules in P.
vulgaris L., but synthesizes indoleacetic acid (IAA) and siderophores,
and solubilizes phosphate. Other strains from the same study identified
as Serratia sp. were antagonists to the phytopathogens Alternaria solani
and Phytophthora capsici.
In order to understand more about NAB from wild legumes in
Mexico, the objective of this work was to analyze the PGP features of
cultivable bacteria isolated from legume nodules of wild plants in
central and southeastern Mexico and bacteria obtained from P. vulgaris
nodules used as a trap plant grown in soil from the same areas.
2. Materials and methods
2.1. Plants, legume nodules and soil sampling
Soil, plants and legume nodules were collected from Chiapas,
México, Guerrero, Morelos, Veracruz and Tabasco states in México
during April and May 2013 (Fig. 1). Chiapas and Veracruz are the states
with the greatest biological diversity in México, and Mexico State,
Guerrero and Tabasco are states where the Mimosa genus is frequently
found. The legumes collected were identified as Acacia sp. W., Acaciella
sp. B. & R., Arachis sp. L., Crotalaria pumila Ort., Desmodium sp. Desv.,
Mimosa affinis B. L. Rob., Mimosa benthamii J. F. Macbr., Mimosa galeottii
Benth., Mimosa pigra L., Mimosa pudica L., Mimosa quadrivalvis L., Leu-
caena sp. Benth., and Trifolium sp. L. The collected plants belonged to
the families Mimosoideae and Papilionoideae. Thirty-four legume no-
dules were recovered from wild plants and 46 rhizospheric soils sam-
ples were taken from the collection sites (Table S1). The legume no-
dules were transported to the laboratory in tubes containing calcium
Fig. 1. Location sites (indicated with green circles) for collecting legume plants and soil analyzed in this study.
2
Microbiological Research 239 (2020) 126522
chloride to keep them dry and the soils were placed in plastic bags. The
plant specimens were preserved for identification, and the soil was used
for plant-trap experiments and physicochemical tests (see below).
2.2. Bacterial isolation from legume nodules
Five legume nodules were randomly selected from each wild plant
and washed with sterile water 3 times. The legume nodule surface was
sterilized by soaking in ethanol for 30 s, immersed in 10 % commercial
chloride for 10 min and rinsed 5 times with sterile water. The water
from the final rinse was used to verify the sterilization. Finally, the
legume nodules were crushed with a plastic pestle with 40 μl of water.
The nodule suspension was inoculated (15 μl) onto plates with both
yeast extract mannitol (YM) medium containing 5 g of mannitol and
peptone yeast (PY) medium. The plates were incubated at 28 °C for 3–5
days. Isolates with different colony morphologies were selected and
stored in 30 % glycerol at −70 °C.
2.3. Plant-trap experiments
The experiments were carried out by mixing soil with vermiculite
(3:1) and adding the mixture to a 325 mL pot. P. vulgaris var. Negro
Jamapa seeds were disinfected with 10 % commercial chloride for 10
min. The seeds were placed in 15 % agar-water plates and incubated at
30 °C for 72 h. The germinated seeds were planted in the pots and
incubated for 45 days in a greenhouse at 30 °C, 14 h light – 10 h dark.
The legume nodules were recovered and treated as described above for
bacterial isolation. For each soil, two replicates were included.
2.4. Bacterial identification
2.4.1. Bacterial DNA isolation
The strains were grown in YM liquid medium for 2 days at 30 °C.
After the incubation period, cell pellets were obtained by centrifugation
(12 300 X g for 5 min). The DNA was isolated according to Ausubel
et al. (1987).
2.4.2. 16S rRNA gene amplification
The 16S rRNA fragment of selected isolates was amplified using
primers 27F/1492R (Lane, 1991). The PCR conditions consisted of an
initial denaturation cycle (95 °C, 5 min), 30 amplification cycles (95 °C,
1 min; 57 °C, 1 min; 72 °C, 1.5 min), and then a final elongation cycle
(72 °C, 10 min). The PCR fragments were sequenced at Macrogen Inc.
(https://dna.macrogen.com/). Sequences were edited with ChromasPro
2.1.5 (Technelysium Pty Ltd). The identification of the isolates was
carried out with the tool BLASTn from NCBI (https://blast.ncbi.nlm.
nih.gov/Blast.cgi) using the database 16S ribosomal RNA sequences for
Bacteria and Archaea and selecting the option Sequences from type
material. All sequences were deposited in GenBank (Table S2).
E.Y. Tapia-García, et al.
2.4.3. Phylogenetic analysis
Nucleotide 16S rRNA alignments were constructed using the soft-
ware MUSCLE (https://www.ebi.ac.uk/Tools/msa/muscle/). The phy-
logenetic tree was constructed using the Bayesian inference method
with the software BEAST v2.6.0 (Bouckaert et al., 2019). jModeltest
2.1.10 software (Darriba et al., 2012) was used to calculate the ap-
propriate evolution model for the data according to the AIC (Akaike
information criterion) (Akaike, 1974). The GTR + G+I model, with α
= 0.6330 for the gamma distribution and p-inv = 0.3430, was used.
2.5. Plant growth-promoting activities
2.5.1. Free-living nitrogen fixation
Free-living nitrogen fixation was tested by inoculating the bacterial
suspension (100 μl, OD600 = 0.1) in 10 ml vials containing 5 ml of
BMGM medium (Estrada-de los Santos et al., 2001). The cultures were
incubated at 29 °C for 72 h. Ten percent of the culture atmosphere was
replaced with acetylene and incubated for 24 h. The bacterial nitrogen
fixation was tested with an acetylene reduction activity (ARA) assay
using a Clarus 580 gas chromatographer (PerkinElmer). Each isolate
was tested in triplicate.
2.5.2. Phosphate solubilization
The isolates were grown in YM medium for 24–72 h at 29 °C. The
cultures were adjusted to 0.1 (OD600), and 2 μl was inoculated on plates
containing NBRIP medium with tricalcium phosphate [Ca3(PO4)2]
(Nautiyal, 1999). The plates were incubated at 29 °C for 3–5 days. The
solubilization halos were measured, and the result was gathered by
subtracting the size of the colony from the size of the halo. Each isolate
was tested in triplicate.
2.5.3. Siderophore production
The universal chemical assay of chromoazurol S (CAS) agar plates
(Caballero-Mellado et al., 2007) was modified by omitting the deferri-
zation process, and 3 % glucose was added as a carbon source in MM9
medium (Schwyn and Neilands, 1987). The bacterial strains were pre-
viously grown in YM medium for 24–72 h at 29 °C. Then, the culture
was adjusted to 0.1 (OD600), and 2 μl was inoculated in CAS-MM9
medium in triplicate. The yellow halos were measured, and the colony
was subtracted to obtain the result.
2.5.4. Indoleacetic acid production
All isolates were grown in 5 ml YM medium at 28 °C for 24–72 h at
120 rpm. The cultures were adjusted to 0.1 (OD600). An aliquot of 100
μl was inoculated into 5 ml of JP medium broth (Jain and Patriquin,
1985), in triplicate and incubated at 29 °C for 72 h. The cultures were
centrifuged at 12,000 rpm, and the supernatant was used to quantify
the production of IAA with Salkowski solution (1:1) (Glickmann and
Dessaux, 1995). After 30 min of incubation at room temperature in the
dark, the absorbance was measured at OD535. Each culture was com-
pared with a standard curve of pure IAA (Sigma-Aldrich).
2.6. nodC gene amplification and phylogenetic inference
The nodC gene was amplified by PCR as an indirect nodulation test.
For Alpha-proteobacteria, a fragment of 560 bp was amplified with the
primers NodCfor540 TGATYGAYATGGARTAYTGGCT and
NodCrev1160 (Sarita et al., 2005). A 635 bp fragment for Beta-pro-
teobacteria nodC was amplified using the primers nodCfor ACTSATA-
CTYAACGTMGAYTC and nodCrev GMRAAYCCRAGAAATCGAAG
(Bontemps et al., 2010). The PCR conditions consisted of an initial
denaturation cycle (95 °C, 5 min), 30 amplification cycles (95 °C, 1 min;
57 °C, 1 min; 72 °C, 45 s), and then a final elongation cycle (72 °C, 10
min). The nodC sequences were compared to those of several genera
that are known to nodulate legumes. The alignment and phylogenetic
analyses were performed similarly to the 16S rRNA sequences analysis.
3
Microbiological Research 239 (2020) 126522
2.7. Inhibition of bacterial plant pathogens
The ability of the strains to control bacterial phytopathogens was
tested using the double-layer agar technique (Rojas-Rojas et al., 2018;
Chávez-Ramírez et al., 2020). Briefly, bacterial strains (producer
strains) were spotted (2 μl from a bacterial culture adjusted to 0.1
OD600) on PDA (potato dextrose agar) medium and incubated at 30 °C
for 72 h. Next, plates were exposed to chloroform vapor and overlaid
with soft agar medium seeded with the phytopathogen bacteria (in-
dicator or sensitive strain). Antimicrobial activity was reported as an
inhibition zone around the producer strain.
2.8. Soil analysis
The collected soil was tested for pH and water retention capacity. A
mixture of soil and water (1:2/5 w/v) was prepared to determine pH
(Thomas, 1996), resulting slightly acidic, with values of 5.8–6.7. The
water retention capacity was quantified gravimetrically (Gardner,
1986), showing that the soils used in the trap experiments were
132–209 %. The analyses were performed with three replicates.
2.9. Data analysis
Venn diagrams were generated using the Venn tool for
Bioinformatics and Evolutive Genomics, using the server at the
University of Ghent (http://bioinformatics.psb.ugent.be/webtools/
Venn/). For the analysis of PGP features (IAA, siderophore biosynth-
esis and phosphate solubilization), media data were compared using the
post hoc Tukey test p < 0.05 with the Minitab v17.1.0 software.
3. Results
3.1. Bacterial isolation from wild plants and trap plants nodules
There were 257 bacteria isolated from wild legume nodules. These
isolates were derived from the plant genera Mimosa, Desmodium,
Arachis, Leucaena, Acacia and Acaciella. Out of the 46 soils used in the
trap experiments, 37 resulted in the development of bean root nodules.
Among these, more than 20 root nodules were formed with 13 soils.
The number of microorganisms isolated from legume nodules in trap
experiments was 171 and the microorganisms were originated from
soils where Mimosa, Leucaena, Crotalaria, Acacia and P. vulgaris plants
were growing.
3.2. Bacterial identification and phylogenetic inference
The identification of the isolates was carried out by sequencing and
analyzing the 16S rRNA fragment. Several genera were identified,
among which the phyla Proteobacteria was the most common and,
Actinobacteria and Firmicutes were the least common (Table 1, Fig. 2).
Within the Proteobacteria, we identified the genera Ensifer, Labrys and
Rhizobium from the Alpha-proteobacteria, the genera Achromobacter,
Burkholderia, Cupriavidus, Herbaspirillum and Paraburkholderia from the
Beta-proteobacteria and the genera Acinetobacter, Aeromonas, Dyella,
Enterobacter, Kosakonia, Moraxella, Pseudomonas, Stenotrophomonas and
Pseudoxhanthomonas from the Gamma-proteobacteria. Within the Ac-
tinobacteria, the genus identified were Brevibacterium, Microbacterium
and Pseudarthrobacter and within the Firmicutes Bacillus, Brevibacillus
and Marinococcus. The genus Pseudomonas was the most common
among the microorganisms isolated from wild legume and trap plant
nodules, and Rhizobium, Cupriavidus and Paraburkholderia were the
most common among the genera recovered from trap plant nodules
after Pseudomonas (Figs. 2 and 3). The phylogenetic analysis from
Alpha-proteobacteria (Fig. S1) showed a strain belonging to the genus
Labrys close to Labrys okinawensis/Labrys monachus (98.35 % similarity)
and a strain from the genus Ensifer close to Ensifer adherens (99.77 %
E.Y. Tapia-García, et al. Microbiological Research 239 (2020) 126522

Table 1
Bacterial species isolated from wild legume nodules in Mexico and nodules from Phaseolus vulgaris used as a trap plant.

(continued on next page)

4
E.Y. Tapia-García, et al. Microbiological Research 239 (2020) 126522

Table 1 (continued)

IAA, indoleacetic acid. For IAA, phosphate solubilization and siderophore production, the asterisk means significative difference compared to the control (above the
control) (P < 0.05) (n = 3). The statistical analysis was performed independently for each plant growth promotion feature tested. In grey are the values that
represent the highest activity.
–, production or activity absent. +, production or activity present. ND, not determined. NI, not identified. The geographical reference for each plant or soil sample is
indicated as: 1N 15°6'4.68” W 92°23'53.447”. 2N 14°57'46.44” W 92°9'20.375”. 3N 14°58'19.308” W 92°9'26.315”. 4N 14°52'30.18” W 92°21'24.768”. 5N 15°5'38.004”
W 92°23'31.239”. 6N 14°52'30.18’’ W 92°21'24.768”. 7N 18°54'20.999” W 99°1'28.999”. 8N 19°0'49” W 98°50'35.998”. 9N 14°58'19.308” W 92°9'26.315”. 10N
15°5'38.004” W 92°23'31.239”. 11N 17°54'51.408” W 99°13'0.407”. 12N 17°36'12.096” W 99°31'25.571”. 13N 17°34'12.576” W 99°35'33.504”. 14N 19°0'49” W 98° 50'
35.998”. 15N 18°0'24.264” W 93°19'8.939”. 16N 18°0'24.264” W 93°19'8.939”. 17N 18°0'27.54” W 93°19'5.664”. 18N 18°5'17.16” W 93°15'10.368”. 19N 18°13'24.816”
W 93°25'0.803”. 20N 18°29'39.516” W 95°2'35.015”.
£
Inhibition of any of the following phytopathogens: Ralstonia solanacearum LMG 2299T, R. solanacearum LMG 2300, Xanthomonas axonopodis pv phaseoli XHFR-02
and Agrobacterium tumefaciens AGJI-04, see Fig. S7 for more details.

similarity). Moreover, a number of strains identified as Rhizobium were
intermingled with several Rhizobium species. Within the Beta-prote-
bacteria (Fig. S2), a strain was close to Herbaspirillum robiniae, 99.42 %
similarity) and a strain to Achromobacter deleyi (99.49 % similarity).
The strains from the genus Burkholderia belong to the Burkholderia ce-
pacia complex and were close to Burkholderia contaminans (99.63 %
similarity). The Paraburkholderia genus was also present, and the strains
were close to Paraburkholderia caballeronis (100 % similarity), Para-
burkholderia unamae (99.35 % similarity), Paraburkholderia mimosarum
(99.71 % similarity) and Paraburkholderia phymatum (96-97-98.40 %
similarities). The strains identified as Cupriavidus were close to Cu-
priavidus oxalaticus (98.52–99.46 similarity), Cupriavidus taiwnanensis/
Cupriavidus alkaliphilus (98.71–98.56 % similarity) and Cupriavidus
metallidurans (99.07–99.10 % similarity). Within the Gamma-proteo-
bacteria (Fig. S3), a strain was close to Enterobacter cloacae (99.78 %
similarity) and several strains were close to Kosakonia cowanii
(99.47–99.55 % similarity), Aeromonas sanarellii (99.62–99.85 % simi-
larity), Moraxella osloensis (99.0–99.13 % similarity) and Pseudox-
anthomonas indica (99.11 % similarity). Some strains were identified as
Acinetobacter lactucae (99.78 % similarity), Acinetobacter lwoffii (99.85
% similarity) and Acinetobacter johnsonii (99.43 % similarity) and other
strains were related to Dyella marensis (99.93–100 % similarity). The
strains identified as Stenotrophomonas were close to Stenotrophomonas
maltophilia (99.28–99.57 % similarity). Pseudomonas was the genus
with the highest number of isolated strains (Fig. S4) which were close to
Pseudomonas oryzihabitans, Pseudomonas plecoglossicida, Pseudomonas
aeruginosa, Pseudomonas entomophila, Pseudomonas reidholzensis, Pseu-
domonas japonica and Pseudomonas ntritireducens, with similarity values
higher than 99 %. Within the Firmicutes (Fig. S5), one strain was close
to Marinococcus tarijensis (99.50 % similarity). Some strains were
identified as Bacillus aryabhattai (99.93 % similarity), Bacillus altitu-
dinis/Bacillus aerius (99.13 % similarity) and Bacillus drentensis (99.42),

5
E.Y. Tapia-García, et al.
Fig. 2. Frequency of bacterial genera identified from legume nodules. WLN, nodules from wild legumes. TPN, nodules from Phaseolus vulgaris used as trap plant.
and two strains were similar to Brevibacillus nitrificans (98.71–99.93 %).
Within the Actinobacteria (Fig. S6), two strains were identified as
Pseudarthrobacter oxydans (99.33–99.77 % similarity). The strain
CpTa8-2b was related to Microbacterium azadirachtae (99.93 % simi-
larity).
3.3. Characterization of bacterial plant growth promotion activity
All strains were analyzed for the production of metabolites involved
directly in plant growth promotion (PGP), such as IAA, and nitrogen
fixation, and for their indirect PGP activity, such as phosphate solubi-
lization, siderophore synthesis or inhibition of bacterial phytopatho-
gens. Forty-four bacteria isolated from wild legumes had at least one
PGP feature, and 35 bacteria with at least one PGP feature were isolated
from trap-plants, for a total of 79 strains in total (Fig. 4). None of the
strains harbored all the analyzed features. However, among the strains
from wild legume nodules, the most common PGP feature was IAA
production (38 strains) (Table 1, Fig. 4). Most of the strains synthesized
IAA, in amounts from 1.84 to 45.09 μg/ml. However, Rhizobium sp.
LIz42a and Stenotrophomonas sp. LXo13 produced more than 250 μg/ml
of IAA, approximately 4 times the quantity synthesized by Azospirillum
brasilense Sp7 (59.77 μg/ml), which is known as a good IAA producer
and was used as a positive control in this analysis (Table 1). Phosphate
solubilization was less common among the strains; only 9 bacteria ob-
tained from wild legume nodules and 17 from trap-plant nodules ex-
hibited phosphate solubilization (Table 1). The ability to fix nitrogen
was found in only 13 strains, one isolated from wild legume nodules
and 12 from trap-plant nodules. The synthesis of siderophores was
present in many strains (45), among which Kosakonia sp. LIz40b41 and
Pseudomonas sp. LIz40b42 (collected from the plant Desmodium sp.,
from Chiapas) exhibited a halo of 3.8 ± 0.01 cm. This halo was the
largest among the isolates from this study and even larger than that
produced by B. cenocepacia TAtl-371, which was 2.2 cm in diameter
6
Microbiological Research 239 (2020) 126522
(Caballero-Mellado et al., 2007), a result that was confirmed in this
study.
3.4. Nodulation potential in the strains
Two sets of primers were used to amplify nodC, one targeting the
nodC from Alpha-proteobacteria and the other targeting nodC from
Beta-proteobacteria. Among the genera found to have the potential to
form legume nodules were Paraburkholderia, Cupriavidus, Labrys,
Brevibacillus, Burkholderia and Rhizobium (Table 2). Among these, the
Beta-proteobacteria Burkholderia and the Firmicutes Brevibacillus are
not known as bacteria that are able to nodulate. The phylogenetic
analysis of the nodC sequences showed that Burkholderia and Breviba-
cillus clustered with alpha-rhizobia, which may have horizontally
transferred to these two genera (Fig. 5).
3.5. Anti-phytopathogenic bacterial activity
All strains were tested against phytopathogenic bacteria such as
Agrobacterium tumefaciens AGJI-04, Ralstonia solanacearum LMG 2299T
and LMG 2300, and Xanthomonas axonopodis pv. phaseoli XHFR-02.
Nine strains from the genera Dyella, Kosakonia and Stenotrophomonas
were able to inhibit the four phytopathogens used in the analysis (Fig.
S7). The strains LXo13, LEh18 and LTz4a from the genus
Stenotrophomonas were able to inhibit only R. solanacearum LMG 2300
(Fig. S7).
4. Discussion
Many plants have developed in and become specialized for the
different climates in Mexico, including the tropical-wet climate in the
southeastern area of the country (Rzedowski, 1991). This part of
Mexico contains the highest number of plant species, especially in the
E.Y. Tapia-García, et al. Microbiological Research 239 (2020) 126522

Fig. 3. Phylogenetic tree based in the analysis of the 16S rRNA sequence from several taxonomic groups which are close to the nodule associated bacteria isolated in
this study. The analysis was performed with the Bayesian inference applying the model GTR + I+G. The bar represents.

Fig. 4. Venn diagrams showing the isolates with plant growth

promotion (PGP) activity. A) Isolates from wild legume nodules
with any PGP activity (N = 43). B) Isolates from Phaseolus vulgaris
nodules used as a trap plant with PGP activity (N = 26). IAA,
indoleacetic acid production. PS, phosphate solubilization ac-
tivity. NF, nitrogen fixation activity. SP, siderophore synthesis.

7
E.Y. Tapia-García, et al. Microbiological Research 239 (2020) 126522

Table 2 Few studies have analyzed NAB from wild legume nodules growing in
Amplification of nodC in bacteria isolated from legume nodules. Mexico. An exploration of M. pudica nodules collected in Chiapas
Species Strain nodC amplification showed a number of strains from the Enterobacter and Serratia genera
with several PGP activities (Sánchez-Cruz et al., 2019). In addition to
Alpha Beta this study, no other reports are known to exist. Therefore, in the present
proteobacteria proteobacteria study, we explored NAB thriving in the root nodules of legume species
Bacteria isolated from wild legume nodules growing in wild areas in the southern Mexico, where a tropical climate
Paraburkholderia sp. LIz1*1 – + prevails. Nodules were collected from wild legumes belonging to the
Cupriavidus sp. LEh21 – + taxa Acaciella sp., Arachis sp., Desmodium sp., M. pudica, M. quadrivalvis,
Cupriavidus sp. LEh25 – + M. galeottii and Leucaena sp. in Chiapas, Tabasco and Veracruz, states in
Paraburkholderia sp. LEh15*2 – +
Mexico known for their richness in legume species (Rzedowski, 1991),
Paraburkholderia sp. LEh19 – +
Labrys sp. LIt4 – + and from Mexico State, Morelos and Guerrero, where many species
from the genus Mimosa exist (Grether et al., 1996; Bontemps et al.,
Bacteria isolated from Phaseolus vulgaris used as trap plant 2016).
Brevibacillus sp. DeCh30-1a*3 + –
Most of the soils (37 out of 48) used in the plant-trap experiments
Burkholderia sp. CpTa8-5 + –
Cupriavidus sp. AcVe19-1a*4 – +
with P. vulgaris resulted in the formation of nodules. P. vulgaris is known
Cupravidus sp. AcVe19-6a*4 + – as a promiscuous plant that can form nodules with several bacterial
Rhizobium sp. AcVe20-2b*4 – + species, namely, Rhizobium, Ensifer and Paraburkholderia, which belong
Paraburkholderia mimosarum PAS44T – + to different groups in Proteobacteria (Martínez-Romero, 2003;
Paraburkholderia phymatum STM815T + +
Dall’Agnol et al., 2016). The genera isolated in this study with PGP
1, strain isolated from Mimosa sp. nodules (Chiapas, N 14° 58' 19.308" W 92° 9' activity were diverse, and Pseudomonas was frequently found in legume
26.315"). 2, Strain isolated from Leucaena sp. nodules (Chiapas, N 14° 52' 30.18" nodules from both wild and trap plants. This is consistent with other
W 92° 21' 24. 768"). 3, Strain isolated from P. vulgaris grown in soil collected studies in which Pseudomonas was abundant as NAB in legumes (Peix
where Desmodium sp. was growing (Chiapas, N 14º 57’ 46.44” W 92º 9’ et al., 2015). The identification of the isolates was performed with the
20.375”). 4, Strain isolated from P. vulgaris grown in soil collected where Acacia 16S rRNA gene, which in many cases and depending on the genus
sp. was growing (Veracruz, N 18º 29’ 39.516 W 95º 2’ 36.015”). analyzed it lacks resolution for the identification at the species level,
Nd, not determined. therefore, other techniques such as MLSA (multilocus sequence ana-
* The strain didn’t show any plant growth promotion activity, therefore are lysis) or whole genome sequencing is used to identify species or to
not listed in Table 1.
describe novel species (Rosselló-Mora and Amann, 2015; Chun et al.,
2018). In this study, the isolated bacteria were identified at the genus
states of Chiapas, Oaxaca and Veracruz (Rzedowski, 1991). The Faba-
level and when possible at species level. The specific genera found in
ceae family (Leguminoseae) is a taxonomic group that is present
the wild legume nodules were Achromobacter, Brevibacillus, Ensifer,
throughout Mexico. This family is divided into three subfamilies a)
Stenotrophomonas, Pseudoxanthomonas, Kosakonia, Dyella and Labrys,
Caesalpinioideae, b) Mimosoideae and c) Papilionoideae (Kenicer,
and the wild plant containing the largest number of bacterial genera
2005). The genus Mimosa from the subfamily Mimosoideae is found
was M. pudica (6 different genera). M. pudica forms nodules with several
extensively in Mexico, which is the second largest radiation center for
bacterial genera, such as Bradyrhizobium, Paraburkholderia, Cupriavidus
this genus (c.a. 100 species) (Grether et al., 1996; Simon et al., 2011).

Fig. 5. Phylogenetic analysis based in the nodC gene sequence of several species from Paraburkholderia (P), Cupriavidus (C), Rhizobium (R), Ensifer (E), Bradyrhizobium
(B), Mesorhizobium (M), and Azorhizobium caulinodans as an out group. The tree was calculated using the Bayesian method with the evolutive model HKY + G. The
bar represents the nucleotide differences between two sequences. Accession numbers of nodC described in this study are in Table S2.

8
E.Y. Tapia-García, et al.
and Rhizobium (Andrews and Andrews, 2017), but no information has
been reported about NAB in this plant. These associations might help
the plant adapt to different soils, given that M. pudica is widely dis-
tributed in the world (https://www.gbif.org/species/2969284). In no-
dules from trap-plants, the specific genera isolated were Burkholderia,
Aeromonas, Moraxella, Herbaspirillum, Enterobacter and Marinococcus. It
has been reported that due to plant domestication (in this case, that of
P. vulgaris, used as a trap-plant), interaction with endophytes di-
minishes (Muresu et al., 2019), but this was not the case in the present
study; both wild legumes and trap plants contained several NAB. This
result may be due to the use of soil from wild plants rather than soil
from agricultural fields for the trap experiments. Furthermore, the soil
from C. pumila used with bean as the trap-plant produced nodules with
the largest number of bacterial genera (7). Crotalaria belongs to the
Papilionoideae subfamily and is a native legume of Mexico, where it is
called “quelite” or more traditionally “chepil”, “chipil” or “chipilin”. It
is an edible plant that is part of traditional agroecosystems but is cur-
rently underused. Some studies have established that this plant controls
Helicobacter pylori-associated diseases (Gomez-Chang et al., 2018) or
has antioxidant effects (Villa-Ruano et al., 2013). However, there is
little information on bacteria associated with C. pumila, although Rhi-
zobium has been isolated from Crotolaria pumila and Crotolaria mollicula
root nodules in France but not in Mexico (Martínez et al., 1985). No-
tably, this plant species has been understudied but seems to be associate
with a number of bacterial genera. Additionally, other genera in addi-
tion to Pseudomonas were found both in wild legumes and trap plant
nodules, such as Cupriavidus, Acinetobacter, Bacillus, Paraburkholderia,
Pseudarthrobacter and Rhizobium. Many of the genera isolated in this
study have been found previously in legume nodules (Abd-Alla et al.,
2018; Benjelloun et al., 2019; Busby et al., 2016; Cardoso et al., 2018;
De Meyer et al., 2015, 2018; Ndlovu et al., 2013; Noori et al., 2018;
Pandya et al., 2013; Rangel et al., 2017; Torche et al., 2016); however,
to the best of our knowledge, this is the first time that Pseudox-
anthomonas, Aeromonas, Pseudarthrobacter and Marinococcus were found
in legume nodules. This adds information to this study of the bacterial
genera found in legume nodules. However, although in this study many
genera were found residing in the legume nodule, the isolation of
bacteria from this structure might be limited by the culture media used
in any analysis, therefore it would be interesting to study the nodule
microbiome from these understudied legumes in Mexico using high
throughput sequencing methods (Hartman et al., 2017).
The potential capacity of the strains to nodulate was tested by
amplifying the nodC gene. Only a few bacteria were positive for the
amplification of this gene. This suggests that most of the strains isolated
in this study are NAB and not typical symbionts; symbionts can be
uncultured, as has been observed when metagenomic analyses are
performed in legume nodules (Martínez-Hidalgo and Hirsch, 2017).
Nonetheless, it was not surprising to find genera that contain this gene
since, since genera such as Paraburkholderia, Cupriavidus, Rhizobium and
Pseudomonas have been reported to be symbionts (Andrus et al., 2012;
Dall’Agnol et al., 2016; González et al., 2019). It was not expected to
find as potential symbionts some bacteria that are typically not known
to exhibit symbiont activity, namely the Beta-proteobacteria Bur-
kholderia and the Firmicutes Brevibacillus. It is important to clarify the
difference between Burkholderia sensu lato and Burkholderia sensu stricto;
the former has been recently divided into the genera Burkholderia,
Paraburkholderia, Caballeronia, Robbsia, Mycetohabitans, Trinickia and
Pararobbsia (Estrada-de los Santos et al., 2018; Lin et al., 2019), and the
latter contains the Burkholderia cepacia complex (Bcc), the Burkholderia
pseudomallei group and a few plant pathogenic species (Rojas-Rojas
et al., 2018). The ability to nodulate has been identified in a number of
species of Paraburkholderia and Trinickia (Estrada-de los Santos et al.,
2018), but not specifically in Burkholderia. The species Burkholderia
vietnamiensis, Burkholderia contaminans and Burkholderia lata are free-
living nitrogen fixers (Gillis et al., 1995; Da Silva et al., 2012) but are
not recognized as nodulating legumes; B. contaminans and B. lata have
9
Microbiological Research 239 (2020) 126522
been isolated from P. vulgaris and Pithecellobium sp. nodules, respec-
tively, but both were uncapable to nodulate siratro and common bean
(Da Silva et al., 2016). Burkholderia sp. CpTa8-5, isolated in this study,
was positive for the amplification of nodC; it belongs to the Bcc, and the
closest species are Burkholderia seminalis/Burkholderia territorii (99.78
%), B. cepacia (99.71 %), and many other Burkholderia species. It is
common to find high 16S rRNA similarity among the species of this
group. A closer phylogenetic analysis comparing the 16S rRNA se-
quence and the atpD gene of Bcc species, the strains CpTa8-5 and
AcTa6-5 showed that both are not related to any Burkholderia species
(data not shown). In the near future, we will analyze the genome se-
quence and test the strain CpTa8-5 in nodulation experiments to de-
termine whether it is a novel Burkholderia species with the ability to
nodulate.
Not much is known about gram-positive bacteria with the ability to
nodulate plant legumes (Ampomah and Huss-Danell, 2011), although
many have been found to be NAB. The Firmicutes Brevibacillus sp.
DeCh30-1a tested positive for the amplification of nodC genes with
Alpha-proteobacteria primers. This is interesting, but more information
needs to be collected to learn about the possible nodulation activity of
this microorganism, such as genome sequencing to identify the genes
involved in nodulation and performing inoculation experiments in
beans and with plants where the soil was collected for the trap plant
experiments, such as Desmodium sp., to determine their ability to no-
dulate.
Among the species found in this study, some were interesting to us,
such as Labrys sp. LIt4, this strain was close to L. neptuniae and was
isolated from the root nodules of Acaciella sp. This strain was positive
for the amplification of nodC, and it produces IAA and fixes nitrogen as
a free-living bacterium. Labrys is not widely recognized as a plant no-
dulating bacteria; a strain identified as Labrys monachus performed ef-
fective symbiosis with Crotalaria spectabilis, but the genes involved in
this activity were not analyzed (Rangel et al., 2017). Therefore, it will
be interesting to more deeply study this bacterium in relation to its
formation of legume nodules in order to show that this genus might be
further involved in symbiosis.
Another species found in this study was P. caballeronis, described as
a nitrogen-fixing bacterium isolated from the rhizosphere of tomato
cultivated in Mexico State and with the ability to effectively nodulate P.
vulgaris (Martínez-Aguilar et al., 2013). However, when the genome
sequence of the type strain was analyzed, no nodulation genes were
found and the ability to nodulate was lost (Rojas-Rojas et al., 2017). In
this study, several P. caballeronis strains were isolated from the nodules
on bean cultivated in soil from Leucaena sp. (Chiapas and Mexico State),
M. benthamii (Guerrero), C. pumila (Tabasco) and Acacia sp. (Veracruz).
None of these strains harbored the nodC gene; the genome of strain
MbGu45-6, also isolated in this study and available from the Joint
Genome Institute (JGI) as NZu45-6, lacks nodulation genes, like the
other P. caballeronis strains available at JGI that we provided for the
GEBA Project (Genomic Encyclopedia of Bacteria and Archaea, JGI-
DOE-USA). It seems that this species may have had nodulation genes
that were lost due to subcultivation in rich culture media or for another
unidentified reason. The role of P. caballeronis as a NAB is interesting to
study because this species was isolated from the soils used in the trap
experiments from several regions of Mexico but not in nodules from
wild legumes collected in the same locations. Apparently, this species
may not be very competent at forming legume nodules in plants other
than P. vulgaris. Currently, this species has been isolated in Mexico but
not in any other country.
Cupriavidus, another noteworthy genus, was found in this study. We
previously reported that C. alkaliphilus, C. plantarum and other
Cupriavidus sp. from the northern Mexico (Tamaulipas state) are asso-
ciated with the rhizosphere of several non-legume plants (Estrada-de los
Santos et al., 2011, 2012, 2014), and from soil and sediment collected
in two contaminated sites in Michoacán state (unpublished data).
However, there are no reports of Cupriavidus species found in legumes
E.Y. Tapia-García, et al.
in Mexico. In this study, Cupriavidus sp. was isolated from wild legume
nodules from Chiapas and a strain from trap-plant cultivated with soil
from Acacia sp. collected in Veracruz. The strains were close to C. al-
kaliphilus (AcVe19-1a, AcVe19-6a, AcVe19-6b and AcVe19-1b), C. ox-
alaticus (LEh21 and LEh25) and C. metallidurans (LEh25b, LXo12c and
LXo12a). From these strains, LEh21, LEh25, AcVe19-1a and AcVe19-6a
were positive for the amplification of nodC. However, it remains to be
confirmed whether the strains are the previously described Cupriavidus
species or whether they represent novel species and symbionts. Nodu-
lation in Cupriavidus has not been shown before in C. alkaliphilus or in C.
oxalaticus. A group of C. alkaliphilus-like strains were found as sym-
bionts from Mimosa spp. growing in alkaline soils on Christmas Island,
Western Australia, but the taxonomy of these strains needs to be re-
viewed in order to confirm whether they belong to C. alkaliphilus or to a
novel Cupriavidus species (De Meyer et al., 2018).
The phylogenetic analysis of nodC sequences showed that the strains
identified as Paraburkholderia, Cupriavidus and Labrys clustered with the
nodC of these genera. However, Burkholderia and Brevibacillus nodC
were placed among Rhizobium species. Since Burkholderia and
Brevibacillus are not known as nodulating bacteria, it is possible that
these strains may have acquired the symbiotic characteristic through
the lateral transfer of symbiotic genes from alpha-rhizobia.
All bacteria recovered from legume nodules were tested for PGP
activity. Rhizobia and NAB might act together as a community in the
nodule to promote plant growth, especially under environmental stress
(Martínez-Hidalgo and Hirsch, 2017). Among the activities tested, the
synthesis of IAA was common among the isolates, with Steno-
trophomonas sp. LXo13 and Rhizobium sp. Liz42a as the greatest pro-
ducers, synthesizing even more than A. brasilense, a typical IAA pro-
ducer. The role of IAA in NAB may be related to improved nodulation
and plant growth when an NAB IAA producer strain is combined with a
symbiotic bacterium (Kumawat et al., 2019). It is possible that the IAA
functions as a signal in bacteria-bacteria communication (quorum
sensing) and between plant-bacteria communication (Spaepen et al.,
2007). The second most common activity was the production of side-
rophores. Kosakonia sp. LIz40b41 and Pseudomonas sp. LIz40b2, were
the greatest siderophore producers. Siderophores are molecules that
bind Fe for leghemoglobin and help to increase the enzymatic activity,
and the water and mineral uptake of the plant (Ferchichi et al., 2019).
Moreover, siderophores can protect plants from pathogens (Martínez-
Hidalgo and Hirsch, 2017). It is possible that some of the siderophore
producer strains isolated in this study exhibit this activity. Another PGP
feature tested was phosphate solubilization. Kosakonia sp. LIz40b41
produced large solubilization halos, twice as larger as those produced
by P. tropica Ppe8, a species known for this activity (Caballero-Mellado
et al., 2007). It has been observed that co-inoculation of phosphate-
solubilizing bacteria with rhizobia increases nodulation, nitrogen fixa-
tion and plant growth (Yasmeen and Bano, 2014). Phosphorus is im-
portant for nitrogen fixation because this activity requires large
amounts of energy in the form of ATP (Pérez‐Fernández et al., 2017;
Stevens et al., 2019). The diversity of NAB found in this study and the
PGP activities performed by these bacteria may represent possible in-
teractions occurring in the nodule among bacteria and with rhizobia.
5. Concluding remarks
This study revealed the presence of NAB belonging to Alpha-, Beta-
and Gamma proteobacteria, Firmicutes and Actinobacteria in un-
explored legumes in Mexico. The majority of the strains were capable of
performing at least one PGP activity that could assist nodule develop-
ment by symbiotic bacteria. New bacterial genera were reported as
NAB, such as Pseudoxanthomonas, Aeromonas, Pseudarthrobacter and
Marinococcus, and potential new symbionts such as Burkholderia from
the B. cepacia complex and strains belonging to the Firmicutes
Brevibacillus were observed. This shows that many bacteria remain
undiscovered in unexplored plants and soils, and they may perform
10
Microbiological Research 239 (2020) 126522
activities that are important for promoting the plant growth.
Funding
This work was partially support by Instituto Politécnico Nacional,
Secretaría de Investigación y Posgrado (México) with the projects:
SIP2016-0077, SIP2017-0492, SIP2018-0117 and SIP2019-6674
awarded to PES.
CRediT authorship contribution statement
Erika Yanet Tapia-García: Conceptualization, Methodology,
Validation, Formal analysis, Investigation, Data curation, Writing -
original draft, Visualization. Verónica Hernández-Trejo:
Methodology, Validation, Investigation. Joseph Guevara-Luna:
Formal analysis. Fernando Uriel Rojas-Rojas: Methodology. Ivan
Arroyo-Herrera: Methodology. Georgina Meza-Radilla:
Methodology, Formal analysis. María Soledad Vásquez-Murrieta:
Funding acquisition, Writing - review & editing. Paulina Estrada-de
los Santos: Conceptualization, Validation, Formal analysis,
Investigation, Resources, Data curation, Writing - original draft, Writing
- review & editing, Visualization, Supervision, Project administration,
Funding acquisition.
Declaration of Competing Interest
The authors declare no conflict of interest.
Acknowledgements
We thank Dr. Carlos Hugo Avendaño Arrazate (INIFAP-Rosario
Izapa) for the logistical support in collecting legumes in Chiapas. We
are grateful to MSc Angélica Martínez Bernal (UAM-Iztapalapa) for
taxonomic identification of the legumes. We thank MSc. Tania Zacaria
Vital for helping to collect plant samples in Guerrero. We are grateful to
Angel Adalberto Cruz Alonzo for technical support. EYTG, JGL, FURR,
IAH, and GMR were recipients of a Consejo Nacional de Ciencia y
Tecnología. fellowship. EYTG, VHT, FURR and GMR were awarded a
BEIFI-IPN fellowship. MSVM and PES thank EDI, COFAA and SNI for
support.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.micres.2020.126522.
References
Abd-Alla, M.H., Bashandy, S.R., Nafady, N.A., Hassan, A.A., 2018. Enhancement of exo-
polysaccharide production by Stenotrophomonas maltophilia and Brevibacillus para-
brevis isolated from root nodules of Cicer arietinum L. and Vigna unguiculata L. (Walp.)
plants. Rend. Fis. Acc. Lincei 29, 117–129.
Akaike, H., 1974. A New Look at the Statistical Model of Identification. Selected Papers of
Hirotugu Akaike Springer, New York, NY, pp. 215–222.
Ampomah, O.Y., Huss-Danell, K., 2011. Genetic diversity of root nodule bacteria nodu-
lating Lotus corniculatus and Anthyllis vulneraria in Sweden. Syst. Appl. Microbiol. 34,
267–275.
Andrews, M., Andrews, M.E., 2017. Specificity in legume-rhizobia symbioses. Int. J. Mol.
Sci. 18, 705.
Andrus, A.D., Andam, C., Parker, M.A., 2012. American origin of Cupriavidus bacteria
associated with invasive Mimosa legumes in the Philippines. FEMS Microbiol. Ecol.
80, 747–750.
Ausubel, F.M., Brent, R., Kingston, R.E., More, D.D., Seidman, J.G., Smith, J.A., Struhl, K.,
1987. Current Protocols in Molecular Biology. John Wiley & Sons, Inc., New
York, N.Y.
Avelar, F.P.A., Bomfeti, C.A., Soares, B.L., de Souza, M.F.M., 2012. Efficient nitrogen-
fixing Rhizobium strains isolated from Amazonian soils are highly tolerant to acidity
and aluminum. World J. Microbiol. Biotechnol. 28, 1947–1959.
Benjelloun, I., Thami-Alami, I., Douira, A., Udupa, S.M., 2019. Phenotypic and genotypic
diversity among symbiotic and non-symbiotic bacteria present in chickpea nodules in
Morocco. Front. Microbiol. 10, 1885.
E.Y. Tapia-García, et al.
Bontemps, C., Elliott, G.N., Simon, M.F., Dos Reis Junior, F.B., Gross, E., Lawton, Nicolau,
E.N., Loureiro, M.D.F., De Faria, S.M., Sprent, J.I., James, E.K., Young, P.W., 2010.
Burkholderia species are ancient symbionts of legumes. Mol. Ecol. 19, 44–52.
Bontemps, C., Rogel, M.A., Wiechmann, A., Mussabekova, A., Moody, S., Simon, M.F.,
Moulin, L., Elliot, G.N., Lacercat-Didier, L., Dasilva, C., Grether, R., Camargo-Ricalde,
S.L., Chen, W., Sprent, J.I., Martínez-Romero, E., Young, J.P.W., James, E.K., 2016.
Endemic Mimosa species from Mexico prefer alphaproteobacterial rhizobial sym-
bionts. New Phytol. 209, 319–333.
Bouckaert, R., Vaughan, T.G., Barido-Sottani, J., Duchêne, S., Fourment, M.,
Gavryushkina, A., Heled, J., Jones, G., Kühnet, D., De Maio, N., Matschiner, M.,
Mendes, F.K., Müller, N.F., Ogilvie, H.A., du Plessis, L., Popinga, A., Rambaut, A.,
Rasmussen, D., Siver, I., Suchard, M.A., Wu, C., Xie, D., Zhang, C., Stadler, T.,
Drummond, A.J., 2019. BEAST 2.5: an advanced software platform for Bayesian
evolutionary analysis. PLoS Comput. Biol. 15, 1–28.
Busby, R.R., Rodriguez, G., Gebhart, D.L., Yannarell, A.C., 2016. Native Lespedeza species
harbor greater non-rhizobial bacterial diversity in root nodules compared to the
coexisting invader, L. cuneate. Plant Soil 401, 427–436.
Caballero-Mellado, J., Onofre-Lemus, J., Estrada-de los Santos, P., Martínez-Aguilar, L.,
2007. The tomato rhizosphere, an enviroment rich in nitrogen fixing Burkholderia
species with capabilities of interest for agriculture and bioremediation. Appl.
Environ. Microb. 73, 5308–5319.
Cardoso, P., Alves, A., Silveira, P., Sá, C., Fidalgo, C., Freitas, R., Figueira, E., 2018.
Bacteria from nodules of wild legume species: phylogenetic diversity, plant growth
promotion abilities and osmotolerance. Sci. Total Environ. 645, 1094–1102.
Chávez-Ramírez, B., Kerber-Díaz, J.C., Acoltzi-Conde, M.C., Ibarra, J.A., Vásquez-
Murrieta, M.S., Estrada-de los Santos, P., 2020. Inhibition of Rhizoctonia solani RhCh-
14 and Pythium ultimum PyFr-14 by Paenibacillus polymyxa NMA1017 and
Burkholderia cenocepacia CACua-24: a proposal for biocontrol of phytopathogenic
fungi. Microbiol. Res. 230, 126347. https://doi.org/10.1016/j.micres.2019.126347.
Chun, J., Oren, A., Ventosa, A., Christensen, H., Ruiz Arahal, D., da Costa, M.S., Rooney,
A.P., Yi, H., De Meyer, S., Trujillo, M.E., 2018. Proposed minimal standards for the
use of genoma data for the taxonomy of prokaryotes. Int. J. Syst. Evol. Microbiol. 68,
461–466. https://doi.org/10.1099/ijsem.0.002516.
Clua, J., Roda, C., Zanetti, M., Blanco, F., 2018. Compatibility between legumes and
rhizobia for the establishment of a successful nitrogen-fixing symbiosis. Genes 9, 125.
https://doi.org/10.1128/AEM.71.11.7461-7471.2005.
Cordeiro, A.B., Ribeiro, R.A., Ferraz Helene, L.C., Hungría, M., 2017. Rhizobium esper-
anzae sp. nov., a N 2 -fixing root symbiont of Phaseolus vulgaris from Mexican soils.
Int. J. Syst. Evol. Microbiol. 67, 3937–3945.
Da Silva, K., de Souza Cassetari, A., Lima, A.S., De Brandt, E., Pinnock, E., Vandamme, P.,
de Souza Moreira, F.M., 2012. Diazotrophic Burkholderia species isolated from the
Amazon region exhibit phenotypical, functional and genetic diversity. Syst. Appl.
Microbiol. 35, 253–262.
Da Silva Chaves, J., Baraúna, A.C., Mosqueira, C.A., Gianluppi, V., Zilli, J.É., Da Silva, K.,
2016. Stylosanthes spp. from Amazon savanna harbour diverse and potentially ef-
fective rhizobia. Appl. Soil. Ecol. 108, 54–61.
Dall’Agnol, R.F., Plotegher, F., Souza, R.C., Mendes, I.C., dos Reis Junior, F.B., Béna, G.,
Moulin, L., Hungria, M., 2016. Paraburkholderia nodosa is the main N2-fixing species
trapped by promiscuous common bean (Phaseolus vulgaris L.) in the Brazilian
‘Cerradão’. FEMS Microbiol. Ecol. 92, 1–13.
Darriba, D., Taboada, G.L., Doallo, R., Posada, D., 2012. jModelTest 2: more models, new
heuristics and parallel computing. Nat. Methods 98, 772.
De Meyer, S.E., De Beuf, K., Vekeman, B., Willems, A., 2015. A large diversity of non-
rhizobial endophytes found in legume root nodules in Flanders (Belgium). Soil Biol.
Biochem. 83, 1–11.
De Meyer, S.E., Cnockaert, M., Moulin, L., Howieson, J.G., Vandamme, P., 2018.
Symbiotic and non-symbiotic Paraburkholderia isolated from South African Lebeckia
ambigua root nodules and the description of Paraburkholderia fynbosensis sp. nov. Int.
J. Syst. Evol. Microbiol. 68, 2607–2614.
Estrada-de los Santos, P., Bustillos-Cristales, R., Caballero-Mellado, J., 2001. Burkholderia,
a genus rich in plant-associated nitrogen fixers with wide environmental and geo-
graphic distribution. Appl. Environ. Microbiol. 67, 2790–2798.
Estrada-de los Santos, P., Martínez-Aguilar, L., López-Lara, I.M., Caballero-Mellado, J.,
2012. Cupriavidus alkaliphilus sp. nov., a new species associated with agricultural
plants that grow in alkaline soils. Syst. Appl. Microbiol. 35, 310–314.
Estrada-de los Santos, P., Palmer, M., Chávez-Ramírez, B., Beukes, C., Steenkamp, E.,
Briscoe, L., Khan, N., Maluk, M., Lafos, M., Humm, E., Arrabit, M., Cook, M., Gross,
E., Simon, M.F., Dos Reis Junior, F.B., Whitman, W.B., Shapiro, N., Poole, S.P.,
Hirsch, A.M., Venter, S.N., James, E.K., 2018. Whole genome analyses suggest that
Burkholderia sensu lato contains two additional novel genera (Mycetohabitans gen.
nov., and Trinickia gen. nov.): implications for the evolution of diazotrophy and
nodulation in the Burkholderiaceae. Genes 9, 389.
Estrada-de Los Santos, P., Vacaseydel-Aceves, N.B., Martínez-Aguilar, L., Cruz-Hernández,
M.A., Mendoza-Herrera, A., Caballero-Mellado, J., 2011. Cupriavidus and
Burkholderia species associated with agricultural plants that grow in alkaline soils. J.
Microbiol. 49, 867–876.
Estrada-de Los Santos, P., Solano-Rodríguez, R., Matsumura-Paz, L.T., Vásquez-Murrieta,
M.S., Martínez-Aguilar, L., 2014. Cupriavidus plantarum sp. nov., a plant-associated
species. Arch. Microbiol. 196, 811–817.
Ferchichi, N., Toukabri, W., Boularess, M., Smaoui, A., Mhamdi, R., Trabelsi, D., 2019.
Isolation, identification and plant growth promotion ability of endophytic bacteria
associated with lupine root nodule grown in Tunisian soil. Arch. Microbiol. 201,
1333–1349.
Gardner, W.H., 1986. Water content. In: Klute, A. (Ed.), Methods of Soil Analysis, Part II.
Physical and Mineralogical Methods, 2nd ed. Am. Soc. Agron., Madison, pp. 493–544
Agronomy Monograph.
11
Microbiological Research 239 (2020) 126522
Gillis, M., Van Van, T., Bardin, R., Goor, M., Hebbar, P., Willems, A., Segers, P., Kersters,
K., Heulin, T., Fernandez, M.P., 1995. Polyphasic taxonomy in the genus Burkholderia
leading to an emended description of the genus and proposition of Burkholderia
vietnamiensis sp. nov. for N2-fixing isolates from rice in Vietnam. Int. J. Syst. Evol.
Microbiol. 45, 274–289.
Glickmann, E., Dessaux, Y., 1995. A critical examination of the specificity of the
Salkowski reagent for indolic compounds produced by phytopathogenic bacteria.
Appl. Environ. Microbiol. 61, 793–796.
Gomez-Chang, E., Uribe-Estanislao, G.V., Martínez-Martínez, M., Gálvez-Mariscal, A.,
Romero, I., 2018. Anti-Helicobacter pylori potential of three edible plants known as
Quelites in Mexico. J. Med. Food 21, 1150–1157.
González, A.H., Morales Londoño, D., Pille da Silva, E., Nascimento, F.X.I., de Souza, L.F.,
da Silva, B.G., Canei, A.D., de Armas, R.D., Giachini, A.J., Soares, C.R.F.S., 2019.
Bradyrhizobium and Pseudomonas strains obtained from coal‐mining areas nodulate
and promote the growth of Calopogonium muconoides plants used in the reclamation
of degraded areas. J. Appl. Microbiol. 126, 523–533.
Grether, R., Camargo, S.L., Martínez, A., 1996. Especies del género Mimosa
(Leguminosae) presentes en México. Bot. Sci. 58, 149–152.
Hartman, K., van der Heijden, M.G.A., Roussely-Provent, V., Walser, J.C., Schlaeppi, K.,
2017. Deciphering composition and function of the root microbiome of a legume
plant. Microbiome 5, 2. https://doi.org/10.1186/s40168-016-0220-z.
Ibáñez, F., Tonelli, M.L., Muñoz, V., Figueredo, M.S., Fabra, A., 2017. Bacterial en-
dophytes of plants: diversity, invasion mechanisms and effects on the host. In:
Maheswary, D.K. (Ed.), Endophytes: Biology and Biotechnology, Sustainable
Development and Biodiversity. Springer International Publishing, pp. 25–40.
Jain, D.K., Patriquin, D.G., 1985. Characterization of a substance produced by
Azospirillum which causes branching of wheat root hairs. Can. J. Microbiol. 31,
206–210.
Kenicer, G., 2005. In: In: Lewis, G., Schrire, B., MacKinder, B., Lock, M. (Eds.), Legumes of
the World 62. Royal Botanic Gardens, Kew. Edinburgh J. Bot., pp. 195–196.
Kumawat, K.C., Sharma, P., Sirari, A., Singh, I., Gill, B.S., Singh, U., Saharan, K., 2019.
Synergism of Pseudomonas aeruginosa (LSE-2) nodule endophyte with Bradyrhizobium
sp. (LSBR-3) for improving plant growth, nutrient acquisition and soil health in
soybean. World J. Microbiol. Biotechnol. 35, 47.
Lane, D.J., 1991. 16S/23S rRNA sequencing. In: Stackebrandt, E., Goodfellow, M. (Eds.),
Nucleic Acid Techniques in Bacterial Systematics. John Wiley and Sons, New York,
pp. 115–175.
Lin, Q.H., Lv, Y.Y., Gao, Z.H., Qiu, L.H., 2019. Pararobbsia silviterrae gen. nov., sp. nov.,
isolated from forest soil and reclassification of Burkholderia alpina as Pararobbsia al-
pina comb. nov. Int. J. Syst. Evol. Microbiol. https://doi.org/10.1099/ijsem.0.
003932.
López-López, A., Rogel, M.A., Ormeno-Orrillo, E., Martínez-Romero, J., Martínez-Romero,
E., 2010. Phaseolus vulgaris seed-borne endophytic community with novel bacterial
species such as Rhizobium endophyticum sp. nov. Syst. Appl. Microbiol. 33, 322–327.
López-López, A., Rogel-Hernández, M.A., Barois, I., Ortiz-Ceballos, A.I., Ormeño-Orrillo,
E., Martínez-Romero, E., 2012. Rhizobium grahamii sp. nov., from nodules of Dalea
leporina, Leucaena leucocephala and Clitoria ternatea, and Rhizobium mesoamer-
icanum sp. nov. from nodules of Phaseolus vulgaris, sirtao, cowpea and Mimosa pu-
dica. Int. J. Syst. Evol. Microbiol. 62, 2264–2271.
Martínez, E., Pardo, M.A., Palacios, R., Miguel, A.C., 1985. Reiteration of nitrogen fixa-
tion gene sequences and specificity of Rhizobium in nodulation and nitrogen fixation
in Phaseolus vulgaris. Microbiology 131, 1779–1786.
Martínez-Aguilar, L., Salazar-Salazar, C., Méndez, R.D., Caballero-Mellado, J., Hirsch,
A.M., Vásquez-Murrieta, M.S., Estrada-de Los Santos, P., 2013. Burkholderia ca-
balleronis sp. nov., a nitrogen fixing species isolated from tomato (Lycopersicon es-
culentum) with the ability to effectively nodulate Phaseolus vulgaris. Antonie van
Leeuwenhoek 104, 1063–1071.
Martínez-Hidalgo, P., Hirsch, A.M., 2017. The nodule microbiome: N2-fixing rhizobia do
not live alone. Phytobiomes 1, 70–82.
Martínez-Romero, E., 2003. Diversity of Rhizboium-Phaseolus vulgaris symbiosis: over-
view and perspectives. Plant Soil 252, 11–23.
Martínez-Romero, E., Segovia, L., Mercante, F.M., Franco, A.A., Graham, P., Pardo, M.A.,
1991. Rhizobium tropici sp. nov., a novel species nodulating Phaseolus vulgaris L. beans
and Leucaenasp. Trees. Int. J. Syst. Bacteriol. 41, 417–426.
Miranda-Sánchez, F., Rivera, J., Vinuesa, P., 2016. Diversity patterns of Rhizobiaceae
communities inhabiting soils, root surfaces and nodules reveal a strong selection of
rhizobial partners by legumes. Environ. Microbiol. 18, 2375–2391.
Muresu, R., Polone, E., Sulas, L., Baldan, B., Tondello, A., Delogu, G., Cappuccinelli, P.,
Alberghini, S., Benhizia, Y., Benhizia, H., Benguedouar, A., Mori, B., Calamassi, R.,
Dazzo, F.B., Squartini, A., 2008. Coexistence of predominantly nonculturable rhi-
zobia with diverse, endophytic bacterial taxa within nodules of wild legumes. FEMS
Microbiol. Ecol. 63, 383–400.
Muresu, R., Porceddu, A., Sulas, L., Squartini, A., 2019. Nodule-associated microbiome
diversity in wild populations of Sulla coronaria reveals clues on the relative im-
portance of culturable rhizobial symbionts and co-infecting endophytes. Microbiol.
Res. 221, 10–14.
Nautiyal, C.S., 1999. An efficient microbiological growth medium for screening phos-
phate solubilizing microorganisms. FEMS Microbiol. Lett. 170, 265–270.
Ndlovu, J., Richardson, D.M., Wilson, J.R., Le Roux, J.J., 2013. Co‐invasion of South
African ecosystems by an Australian legume and its rhizobial symbionts. J. Biogeogr.
40, 1240–1251.
Noori, F., Etesami, H., Zarini, H.N., Khoshkholgh-Sima, N.A., Salekdeh, G.H., Alishahi, F.,
2018. Mining alfalfa (Medicago sativa L.) nodules for salinity tolerant non-rhizobial
bacteria to improve growth of alfalfa under salinity stress. Ecotoxicol. Environ. Saf.
162, 129–138.
Pandya, M., Naresh Kumar, G., Rajkumar, S., 2013. Invasion of rhizobial infection thread
E.Y. Tapia-García, et al.
by non-rhizobia for colonization of Vigna radiata root nodules. FEMS Microbiol. Lett.
348, 58–65.
Parker, M.A., 2002. Bradyrhizobia from wild Phaseolus, Desmodium, and Macroptilium
species in Northern Mexico. Appl. Environ. Microbiol. 68, 2044–2048.
Peix, A., Ramírez-Bahena, M.H., Velázquez, E., Bedmar, E.J., 2015. Bacterial associations
with legumes. Crit. Rev. Plant Sci. 34, 17–42.
Pérez‐Fernández, M.A., Calvo‐Magro, E., Rodríguez‐Sánchez, J., Valentine, A., 2017.
Differential growth costs and nitrogen fixation in Cytisus multiflorus (L′ Hér.) Sweet
and Cytisus scoparius (L.) are mediated by sources of inorganic N. Plant Biol. 19,
742–748.
Rangel, W.D.M., de Oliveira Longatti, S.M., Ferreira, P.A.A., Bonaldi, D.S., Guimaraes,
A.A., Thijs, S., Weyes, N., Vangrosveld, J., Moreira, F.M., 2017. Leguminosae native
nodulating bacteria from a gold mine As-contaminated soil: multi-resistance to trace
elements, and possible role in plant growth and mineral nutrition. Int. J.
Phytoremediation 19, 925–936.
Rincón-Rosales, R., Lloret, L., Ponce, E., Martínez-Romero, E., 2009. Rhizobia with dif-
ferents symbiotic efficiencies nodulate Acaciella angustissima in Mexico, including
Sinorhizobium chiapanecum sp. nov. which has common symbiotic genes with
Sinorhizobium mexicanum. FEMS Microbiol. Ecol. 67, 103–117.
Rojas-Rojas, F.U., Tapia-García, E.Y., Maymon, M., Humm, E., Huntemann, M., Clum, A.,
Pillay, M., Palaniappan, K., Varghese, N., Mikhailova, N., Stamatis, D., Reddy, T.B.K.,
Markowitz, V., Ivanova, N., Kyrpides, N., Woyke, T., Shapiro, N., Hirsch, A.M.,
Estrada-de los Santos, P., 2017. Draft genome of Paraburkholderia caballeronis TNe-
841T, a free-living, nitrogen-fixing, tomato plant-associated bacterium. Stand.
Genomic Sci. 12, 80.
Rojas-Rojas, F.U., Salazar-Gómez, A., Vargas-Díaz, M.E., Vásquez-Murrieta, M.S., Hirsch,
A.M., De Mot, R., Maarten, G.K., Ghequire, J., Ibarra, A., Estrada-de los Santos, P.,
2018. Broad-spectrum antimicrobial activity by Burkholderia cenocepacia TAtl-371, a
strain isolated from the tomato rhizosphere. Microbiology 164, 1072–1086.
Román-Ponce, B., Zhang, Y.J., Vásquez-Murrieta, M.S., Sui, X.H., Chen, W.F., Padilla,
J.C.A., Wang, E.T., 2016. Rhizobium acidisoli sp. nov., isolated from root nodules of
Phaseolus vulgaris in acid soils. Int. J. Syst. Evol. Microbiol. 66, 398–406.
Rosselló-Mora, R., Amann, R., 2015. Past and future species definitions for Bacteria and
Archaea. Syst. Appl. Microbiol. 38, 209–216. https://doi.org/10.1016/j.syapm.2015.
02.001.
Rzedowski, J., 1991. Diversidad y orígenes de la flora fanerogámica de México. Acta Bot.
Mex. 14, 3–21.
Sánchez-Cruz, R., Vázquez, I.T., Batista-García, R.A., Méndez-Santiago, E.W., del Rayo
Sánchez-Carbente, M., Leija, A., Lira-Ruan, V., Hernández, G., Wong-Villarreal, A.,
Folch-Mallol, J.L., 2019. Isolation and characterization of endophytes from nodules
12
Microbiological Research 239 (2020) 126522
of Mimosa pudica with biotechnological potential. Microbiol. Res. 218, 76–86.
Sarita, S., Sharma, P.K., Priefer, U.B., Prell, J., 2005. Direct amplification of rhizobial
nodC sequences from soil total DNA and comparison to nodC diversity of root nodule
isolates. FEMS Microbiol. Ecol. 54, 1–11.
Schwyn, B., Neilands, J.B., 1987. Universal chemical assay for the detection and de-
termination of siderophores. Anal. Biochem. 160, 47–56.
Segovia, L., Young, J.P., Martínez-Romero, E., 1993. Reclassification of american
Rhizobium leguminosarum biovar phaseoli type I as Rhizboium etli sp. nov. Int. J. Syst.
Bacteriol. 43, 374–377.
Selvakumar, G., Kundu, S., Anand, D.G., Yogesh, S.S., Hari, S.G., 2008. Isolation and
characterization of non-rhizobial plant growth promoting bacteria from nodules of
kudzu (Pueraria thunbergiana) and their effect on wheat seedling growth. Curr.
Microbiol. 56, 134–139.
Simon, M.F., Grether, R., de Queiroz, L.P., Särkinen, T.E., Dutra, V.F., Hughes, C.E., 2011.
The evolutionary history of Mimosa (Leguminosae): toward a phylogeny of the sen-
sitive plants. Am. J. Bot. 98, 1201–1221.
Spaepen, S., Vanderleyden, J., Remans, R., 2007. Indole-3-acetic acid in microbial and
microorganism-plant signaling. FEMS Microbiol. Rev. 31, 425–448.
Stevens, G.G., Pérez-Fernádez, M.A., Morcillo, R.J., Kleinert, A., Hills, P.N., Brand, D.J.,
Steenkamp, T., Valentine, A.J., 2019. Roots and nodules response differently to P
starvation in the Mediterranean-type legume Virgilia divaricata. Front. Plant Sci.
10, 73.
Thomas, G.W., 1996. Soil pH and soil acidity. In: Sparks, D.L. (Ed.), Methods of Soil
Analysis. Chemical Methods. Soil Sci. Soc. of Am., Madison, pp. 475–490.
Torche, A., Benhizia, H., Rosselli, R., Romoli, O., Zanardo, M., Baldan, E., Alberghini, S.,
Tondello, A., Baldan, B., Benguedouar, A., Squartini, A., Benhizia, Y., 2016.
Characterization of bacteria associated with nodules of two endemic legumes of
Algeria, Hedysarum naudinianum and H. perrauderianum. Ann. Microbiol. 64,
1065–1071.
Villa-Ruano, N., Zurita-Vásquez, G.G., Pacheco-Hernández, Y., Betancourt-Jiménez, M.G.,
Cruz-Durán, R., Duque-Bautista, H., 2013. Anti-Iipase and antioxidant properties of
30 medicinal plants used in Oaxaca, México. Biol. Res. 46, 153–160.
Wang, E.T., Rogel, M.A., García de los Santos, A., 1999. Rhizobium etli bv. mimosae, a
novel biovar isolated from Mimosa affinis. Int. J. Syst. Bacteriol. 49, 1479–1491.
Yan, J., Yan, H., Liu, X., Chen, W.F., Zhang, X.X., Verastegui-Valdes, M.M., Wang, E.T.,
Han, X.Z., 2017. Rhizobium hidalgoense sp. nov., a nodule endophytic bacterium of
Phaseolus vulgaris in acid soil. Arch. Microbiol. 199, 97–104.
Yasmeen, S., Bano, A., 2014. Combined effect of phosphate-solubilizing microorganisms,
Rhizobium and Enterobacter on root nodulation and physiology of soybean (Glycine
max L.). Commun. Soil Sci. Plant Anal. 45, 2373–2384.