Applied Soil Ecology 182 (2023) 104731

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Applied Soil Ecology

journal homepage: www.elsevier.com/locate/apsoil

Rhizospheric microbial community in plant species from the

Phaseolus genus
Angela Celis de Almeida Lopes a, Lucas William Mendes b,
Karla Annielle da Silva Bernardo Brito a, Josieli Lima da Silva a, Sandra Mara Barbosa Rocha c,
Jadson Emanuel Lopes Antunes c, Louise Melo de Souza Oliveira c, Vania Maria Maciel Melo d,
Francisca Andrea Silva Oliveira d, Arthur Prudêncio de Araujo Pereira e,
Gérson do Nascimento Costa a, Veronica Brito da Silva a, Regina Lucia Ferreira Gomes a,
Francisco de Alcantara Neto f, Ademir Sergio Ferreira Araujo c, *
a
Plant Genetic Resource Group, Agricultural Science Center, Federal University of Piauí, Teresina, PI, Brazil
b
Center for Nuclear Energy in Agriculture, University of Sao Paulo, Piracicaba, SP, Brazil
c
Soil Microbial Ecology Group, Agricultural Science Center, Federal University of Piauí, Teresina, PI, Brazil
d
Laboratório de Ecologia Microbiana e Biotecnologia, Federal University of Ceará, Fortaleza, CE, Brazil
e
Soil Science Department, Federal University of Ceará, Fortaleza, CE, Brazil
f
Plant Science Department, Agricultural Science Center, Federal University of Piauí, Teresina, PI, Brazil

A R T I C L E I N F O A B S T R A C T

Keywords: The rhizospheric microbial community is affected by plant genera and genotypes. However, the rhizospheric
Plant science effect on the microbial community from distinct plant species belonging to the same genus is poorly understood.
Plant domestication This study hypothesized that the rhizospheric microbial community differs between plant species from the
Bacterial community
Phaseolus genus (i.e. P. acutifolius A. Gray, P. lunatus L., P. vulgaris L., P. microcarpus Mart., and P. filiformis Benth.)
Amplicon sequencing
and that these differences could be related to phylogenetic differences found in these plant species. The
redundancy analysis showed a distinct microbial community mainly when comparing P. lunatus and
P. microcarpus. The microbial richness and diversity varied among plants, where P. lunatus showed the highest
richness and diversity and P. microcarpus the lowest. The abundances of specific microbial phyla varied between
the rhizosphere of Phaseolus species. Actinobacteria was abundant in the rhizosphere of P. acutifolius, P. vulgaris,
and P. filiformis, while Acidobacteria was abundant in the rhizosphere of P. lunatus. This study revealed that each
Phaseolus species recruits a distinct microbial community in the rhizosphere, with the rhizosphere community of
the domesticated species P. lunatus being the most distinct.

1. Introduction In plants, the rhizospheric effect on microbial community differs

The rhizosphere is a soil zone driven by plant roots and their varied
exudates, e.g., sugars and organic acids, which can recruit a variety of
microbes (Canarini et al., 2019). In the rhizosphere, plants communicate
with the microbial community through root exudation (Williams et al.,
2022), mostly attracting beneficial microbes. For instance, plant growth-
promoting microbes can be attracted and contribute to plant growth
(Raaijmakers et al., 2009). Therefore, it is important to assess the mi­
crobial community inhabiting the rhizosphere to better understand how
each plant species can shape this community.
according to plant species (Araujo et al., 2019), genotypes (Sousa et al.,
2020), and their stage of development (Moroenyane et al., 2021).
However, when comparing plant species from the same genus, there is
not enough information about how the microbial community is influ­
enced. As an important genus of plants, Phaseolus belongs to the family
Fabaceae and contains several plant species (Kaplan, 2003), being
economically important mainly to Latin America. Some Phaseolus spe­
cies are domesticated, such as P. acutifolius A. Gray., P. lunatus L., and
P. vulgaris L. (Bitocchi et al., 2017), while there are several wild species,
such as P. microcarpus Mart., and P. filiformis Benth. However, studies

* Corresponding author at: Soil Quality Lab., Agricultural Science Center, Federal University of Piauí, Teresina, PI, Brazil.
E-mail addresses: asfaruaj@yahoo.com.br, ademir@ufpi.edu.br (A.S.F. Araujo).

https://doi.org/10.1016/j.apsoil.2022.104731
Received 15 August 2022; Received in revised form 30 September 2022; Accepted 4 November 2022
Available online 12 November 2022
0929-1393/© 2022 Elsevier B.V. All rights reserved.
A.C.A. Lopes et al.
focusing on the microbial community in the rhizosphere of the Phaseolus
genus are concentrated in P. vulgaris and P. lunatus (Mendes et al.,
2018b, 2018a; Pérez-Jaramillo et al., 2017). For example, Pérez-Jar­
amillo et al. (2017) assessed the microbial community in the rhizosphere
of wild and domesticated P. vulgaris and observed changes in some mi­
crobial phyla, such as a decrease in Bacteroidetes and an increase in
Proteobacteria from wild to domesticated species. In the rhizosphere of
P. lunatus, Sousa et al. (2020) evaluated the bacterial community
comparing bulk soil and rhizosphere of domesticated genotypes and
found an increase in Proteobacteria and Actinobacteria and a decrease in
Firmicutes from bulk soil to the rhizosphere.
Although these previous studies have found distinct microbial com­
munities in the rhizosphere of P. vulgaris and P. lunatus, there is no study
comparing the microbial community in the rhizosphere of some wild
species, such as P. microcarpus and P. filiformis. Genetically, previous
studies have reported that both domesticated P. vulgaris and P. lunatus
present reduced genetic diversity when compared to wild species of
Phaseolus species (Motta-Aldana et al., 2010; Chacón-Sánchez and
Martínez-Castillo, 2017). In addition, a previous study comparing the
phylogeny of the Phaseolus genus classified all known species in two
clades (A and B), where P. microcarpus belongs to clade A, and
P. acutifolius, P. vulgaris, P. filiformis, and P. lunatus belong to clade B
(Delgado-Salinas et al., 2006). Interestingly, P. acutifolius, P. vulgaris,
and P. filiformis are genetically close, while P. lunatus and P. microcarpus
are distant (Delgado-Salinas et al., 2006). These genetic traits presented
by each species can influence the rhizosphere and shape the microbial
community.
On the other hand, plant domestication significantly impacts the
physiology, morphology, and genetics of plants. In general, the allelic
diversity of domesticated plants decreases due to target and non-target
selection (Doebley et al., 2006). However, there is little understanding
Fig. 1. Map showing the Phaseolus species used in this study and their origin and genetic traits. Clades were classified according to Delgado-Salinas et al. (2006).
Rates (substitutions per site per million years) and ages (millions of years) from trnK and ITS/5.8S sequences for the crown clades (Delgado-Salinas et al., 2006). BGP
– Banco Germoplasma Phaseolus; UFPI – Universidade Federal do Piaui.
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of how changes in plant genotypes during domestication influence the
rhizosphere microbiome assembly in plants within the same genus.
Considering the changes in plant genetic and physiological traits during
the domestication process, this study hypothesized that (1) the rhizo­
spheric microbial community differs comparing plant species from the
Phaseolus genus, and (2) there would be differences in the rhizospheric
microbial community of both genetically close and distant Phaseolus
species. To address these hypotheses, we evaluated the rhizospheric
microbial community of five species from the Phaseolus genus, namely
P. acutifolius, P. lunatus, P. vulgaris, P. microcarpus, and P. filiformis
through the sequencing of the 16S rRNA gene.
2. Material and methods
2.1. Greenhouse experiment
This study was conducted in pots containing ~4 kg of soil and placed
in a greenhouse belonging to the Universidade Federal do Piauí, Brazil.
In this location, the average precipitation and temperature are 1200 mm
yr− 1 and 28 ◦ C, respectively. We used a sandy soil (Fluvisol) with the
following chemical characteristics (0–0.2 m layer): pH – 6.4; organic
matter - 9.1 g kg− 1; P – 4.1 mg kg− 1; K – 38.6 mg kg− 1; base saturation
(V) – 62 %. The experimental design consisted of the five plant species
from the Phaseolus genus, namely P. acutifolius, P. lunatus, P. vulgaris,
P. microcarpus, and P. filiformis (Fig. 1). The genotypes from each plant
species were provided by the Genetic Resources Lab. from the Uni­
versidade Federal do Piauí. In this study, we used the phylogenetic data
and some important plant traits obtained from Delgado-Salinas et al.
(2006).
A.C.A. Lopes et al.
2.2. Plant growth and sampling of rhizospheric soil
Six seeds of each plant species were sown per pot (three pots per
genotypes) and, 10 days after germination, one plant was left per pot.
The soil moisture was kept (70 % of field capacity) using sterilized
water. Plants were sampled at the flowering stage for each genotype.
The bulk soil consisted of three soil subsamples (replicates). The soil
adhered to roots (rhizospheric soil) was collected and kept at − 80 ◦ C.
Therefore, a total of 18 soil samples (three bulk soil and fifteen rhizo­
spheric soil) were assessed.
2.3. Microbial community profiling
In each sample, a portion of 0.5 g of soil was used to extract DNA
using the Powerlyzer PowerSoil DNA Isolation Kit (MoBio Laboratories,
Carlsbad, CA, USA), and the quality and concentration of the extracted
DNA were evaluated by a spectrophotometer. The V4 region of the 16S
rRNA gene, spanning bacteria and archaea, was amplified with region-
specific primers (515F/806R) (Caporaso et al., 2011). Each Polymer­
ase Chain Reaction (PCR) used nuclease-free water (12.25 μL), buffer
solution (5.0 μL), Taq polymerase (1.0 unit; concentration of 0.5 μL),
and template DNA (2.0 μL; 10 ng μL− 1). PCR followed 95 ◦ C for 3 min,
with 35 cycles at 98 ◦ C for 20 s, 55 ◦ C for 20 s, 72 ◦ C for 30 s, and 3 min at
72 ◦ C. After indexing, the PCR products were cleaned up using Agen­
court AMPure XP and quantified using the dsDNA BR assay Kit on a
fluorometer. After quantification, the molarity of the pool was deter­
mined and diluted to 2 nM, denatured, and then diluted to a final con­
centration of 8.0 pM with a 20 % PhiX spike for loading into the Illumina
MiSeq sequencing machine (Illumina, San Diego, CA, USA).
The 16S rRNA sequences generated were processed using the soft­
ware QIIME 2 version 2021.11 (Caporaso et al., 2011). The sequences
were demultiplexed and quality control was carried out using DADA2
(Callahan et al., 2016), with the consensus method to remove any
remaining chimeric and low-quality sequences. A total of 2,455,000
high-quality sequences were obtained, being rarefied to 78,300 se­
quences, following the sample with the lowest amount of sequences. The
taxonomic identification was performed using the SILVA database
(v.132) at 97 % of similarity (Quast et al., 2013). The sequences are
deposited at the NCBI Sequence Read Archive under the project number
PRJNA861144.
2.4. Data analysis
We firstly checked the data for normal distribution and homogeneity
of variance using the Shapiro-Wilk and Levene’s tests using the R soft­
ware. Then, we assessed the bacterial and archaeal community structure
of bulk soil and rhizosphere using the Principal Component Analysis
(PCA). We then used Redundancy analysis (RDA) to assess the rhizo­
sphere community structure and its correlation with plant traits. PCA
and RDA analyses were conducted in Canoco 5 (Biometrics, Wagenin­
gen, The Netherlands). The indices of diversity and richness were esti­
mated by OTU level and compared by Tukey (Hammer et al., 2001). The
differential abundance of bacterial groups at the phylum and OTU level
was compared using the Statistical Analysis of Metagenomic Profile
(STAMP) software (Parks and Beiko, 2010). P-values were calculated
using a two-sided Tukey-Kramer test, and corrections were made using
Benjamini-Hochberg’s false discovery rate (Benjamini and Hochberg,
1995). Network analysis was performed to assess the complexity of in­
teractions among microbial taxa in each site. For this, non-random co-
occurrence analysis was carried out using the Python module ‘SparCC’
(Friedman and Alm, 2012). For this, a table of frequency of OTUs was
used for analysis. To decrease the number of correlations, we included
only the OTUs with >50 sequences, which represented >80 % of the
total sequences. For each network, the SparCC correlations were
calculated, selecting the most significant correlations. The nodes in the
reconstructed network represent OTUs, whereas the edges represent
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Applied Soil Ecology 182 (2023) 104731
significantly positive or negative correlations between nodes. The
network visualization and properties measurements were calculated
with the interactive platform Gephi (Bastian et al., 2009). Lastly, the
potential functional profile was assessed through the FAPROTAX 1.2.5
database (Louca et al., 2019). This database maps prokaryotic clades (e.
g. genera or species) to established putative metabolic functions using
information based on functional annotations of cultivated representa­
tives (Merloti et al., 2019). For this, the frequency table of hits of taxa
generated from QIIME at genus level was used as input and converted
into a putative functional table.
3. Results
3.1. Microbial community structure
We used PCA analysis to compare the microbial community structure
among soil and rhizosphere of different Phaseolus species and the first
two axes explained 15.8 % and 9.3 %, respectively, of the total variation
in the microbial community (Fig. 2A). The analysis showed a distinct
microbial community comparing different plant species, mainly when
comparing the rhizospheric microbial community of P. lunatus and
P. microcarpus (PERMANOVA F = 2.594, p = 0.0001). On the other
hand, the rhizosphere of P. acutifolius, P. vulgaris, and P. filiformis pre­
sented a similar microbial community. Interestingly, the soil showed a
similar structure of microbial community to P. filiformis. We then used
RDA analysis to compare the rhizosphere microbial community of each
Phaseolus species and correlate it with plant traits (Fig. 2B). Thus, the
microbial community in the rhizosphere of P. microcarpus correlated
with ITS rate and trnK (age and rate), while the microbial community in
the rhizosphere of P. lunatus clustered with higher values of seed length
and root system.
3.2. Microbial community composition
The most abundant phyla found in the rhizosphere of plants from
Phaseolus species were Actinobacteria (27.9 % of the total sequences),
followed by Proteobacteria (18.3 %), Firmicutes (14.1 %), Chloroflexi
(7.9 %), Acidobacteria (7.7 %), Thaumarchaeota (6.9 %), Planctomy­
cetes (6.3 %), unclassified Bacteria (2.9 %), Verrucomicrobia (1.4 %),
and Bacteroidetes (1.2 %) (Fig. 3A). The proportion of sequences of
specific phyla differed between the rhizosphere of Phaseolus species.
Actinobacteria was abundant in the rhizosphere of P. acutifolius, P. vul­
garis, and P. filiformis, while Acidobacteria was abundant in the rhizo­
sphere of P. lunatus. Interestingly, Cyanobacteria was significantly more
abundant in the rhizosphere of P. microcarpus. The microbial richness
(Fig. 3B) and diversity (Fig. 3C) varied among plant species, where
P. lunatus showed the highest richness and diversity and P. microcarpus
the lowest. We then highlighted the top ten most abundant rhizospheric
microbial OTUs from all samples, which accounted for 32 % of the total
microbial community (Fig. 3D). The most abundant OTUs were affili­
ated to Bacillus, followed by Nitrososphaeraceae, Bacillales, Acid­
obacteria Subgroup 6, and unclassified Bacteria. Interestingly, Bacillus
and Acidobacteria Subgroup 6 were the most abundant in the rhizo­
sphere of P. microcarpus and P. lunatus, respectively, while Bacillales was
abundant in the rhizosphere of P. acutifolius, P. vulgaris, and P. filiformis.
At the OTU level, the analysis showed a total of 386 OTUs with
distinct abundances between the treatments, with the formation of five
distinct clusters. Cluster IV (197 OTUs) and cluster III (68 OTUs) pre­
sented the most differential OTUs, while clusters I, II, and V presented
54, 35, and 32 differential OTUs, respectively (Fig. 4A). Interestingly,
we observed a clear separation between microbial OTUs found in both
clusters IV and III, which were enriched in the rhizosphere of P. lunatus
and P. microcarpus, respectively. Less clearly, microbial OTUs found in
the rhizospheres of P. filiformis, P. acutifolius, and P. vulgaris were
grouped into clusters I, II, and V, respectively. This analysis showed that
P. lunatus presented the most distinct rhizospheric microbial
A.C.A. Lopes et al. Applied Soil Ecology 182 (2023) 104731

Fig. 2. Structure of the bacterial and archaeal communities in bulk soil and rhizosphere of different species of Phaseolus genus, based on the 16S rRNA gene. (A)
Principal component analysis (PCA) including samples from bulk soil and rhizosphere. (B) Redundancy analysis (RDA) of the microbial community and correlation
with plant characteristics. The analysis of permutation (PERMANOVA) is indicated in the upper right of each graph.

Fig. 3. (A) Microbial community composition at phylum level in bulk soil and rhizosphere of different Phaseolus species based on the 16S rRNA gene. (B) Richness
and (C) Shannon’s diversity index measurements of the microbial communities at OTU level. Error bars represent standard deviation of three independent replicates.
Different lower-case letters refer to significant differences between treatments based on Tukey’s HSD test (P < 0.05). (D) The top 10 most abundant microbial OTUs in
the rhizosphere samples.

community. We then compared each treatment with all others to detect
the differential OTUs. The pairwise comparison showed different mi­
crobial groups enriched in each rhizosphere, where the rhizosphere of
P. vulgaris (19 OTUs) enriched OTUs affiliated to Rhizobiaceae (Fig. 4B),
while the rhizosphere of P. acutifolius (54 OTUs) enriched Chloroflexi
KD4–96 and Gaiellales (Fig. 4C). The rhizosphere of P. filiformis (43
OTUs) enriched unclassified_Bacteria and Gaiellales (Fig. 4D), and the
rhizosphere of P. lunatus (151 OTUs) enriched Pyrinomonadaceae
(Fig. 4E). Finally, the rhizosphere of P. microcarpus (36 OTUs) enriched
Pirellulaceae (Fig. 4F), while bulk soil (50 OTUs) harbored a signifi­
cantly higher abundance of sequences of Bacillales (Fig. 4G). Again, this
analysis showed that P. lunatus was the most distinct treatment, with
enrichment of 151 OTUs in its rhizosphere, followed by P. acutifolius (44
OTUs), P. filiformis (43 OTUs), P. microcarpus (36 OTUs), and P. vulgaris
(19 OTUs). Together, this analysis revealed that each Phaseolus species
recruit a distinct microbial community in its rhizosphere, with the
rhizosphere community of the domesticated species P. lunatus being the
most distinct.

4
A.C.A. Lopes et al.
Fig. 4. Microbial community composition in bulk soil and the rhizosphere of different Phaseolus species based on 16S rRNA. (A) Heatmap showing the differential
abundance of OTUs among all treatments. (B-G) Pairwise comparison between each treatment against all others. Only the top 10 most differential OTUs are shown.
The total number of differential OTUs is indicated in the upper of each graph. In all graphs, only significant different OTUs are shown, based on Welch’s t-test with
Benjamini-Hochberg correction (P < 0.05).
3.3. Microbial co-occurrence network
The analysis of the microbial co-occurrence network compared the
complexity of connections between the rhizospheric microbial com­
munities of distinct plant species (Fig. 5; Table S1). Firstly, we observed
an increased complexity from the soil to the rhizosphere. Second, when
compared microbial community in the rhizosphere of each Phaseolus
species, the complexity of connections increased from P. acutifolius
(nodes = 316; edges = 2126), P. microcarpus (nodes = 353; edges =
2525), P. filiformis (nodes = 384; edges = 2531), P. lunatus (nodes = 422;
edges = 3665) to P. vulgaris (nodes = 527; edges = 9453). Again,
P. lunatus was the most distinct species, with the highest complexity,
average degree, and lower number of communities. Also, the proportion
of positive interactions was higher in the rhizosphere than in the soil.
3.4. Functional prediction related to the nitrogen cycle
We found six predicted functions with a different abundance ac­
cording to the treatments (Fig. 6). In general, the species P. lunatus and
P. microcarpus exhibited differences compared to other species, with the
latter presenting a lower abundance of sequences affiliated to nitrogen
functions. On the other hand, P. lunatus presented a higher abundance of
sequences affiliated to nitrification, aerobic ammonia oxidation, aerobic
nitrite oxidation, and nitrate reduction.
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Applied Soil Ecology 182 (2023) 104731
4. Discussion
4.1. The rhizosphere microbial community structure
This study assessed the microbial communities in the rhizosphere of
five plant species belonging to the Phaseolus genus, which present
phylogenetic differences (Delgado-Salinas et al., 2006). In line with
hypothesis (1), the rhizospheric microbial community differed among
the different plant species. It confirms that different plant species, even
belonging to the same genus, recruit a distinct microbial community in
the rhizosphere. In addition, the results of the microbial community
structure in the rhizosphere confirm the hypothesis (2) that plant species
phylogenetically close, such as P. filiformis, P. acutifolius, and P. vulgaris,
present a more similar microbial community, while plant species
phylogenetically different, i.e., P. lunatus and P. microcarpus, showed a
more distinct microbial community. According to Delgado-Salinas et al.
(2006), P. filiformis, P. acutifolius, and P. vulgaris present >85 % of
phylogenetic similarity, which could explain the recruitment of a similar
rhizospheric microbial community. In contrast, P. microcarpus and
P. lunatus are classified into different clades (A and B, respectively),
presenting a higher phylogenetic dissimilarity (Delgado-Salinas et al.,
2006). Interestingly, the rhizosphere community presented the same
pattern of similarity, following the phylogenetic traits, as can be seen in
the PCA analysis. Also, the RDA analysis showed that the microbial
community in the rhizosphere of P. microcarpus correlated with ITS
phylogeny and trnK gene, being different from other plant species of
Phaseolus, as observed by Fonsêca and Pedrosa-Harand (2013)
comparing the genetic phylogeny of P. microcarpus, P. vulgaris, and
P. lunatus. The PCA results also showed the soil presenting a similar
A.C.A. Lopes et al. Applied Soil Ecology 182 (2023) 104731

Fig. 5. Network co-occurrence analysis of the microbial communities in bulk soil and rhizosphere of different Phaseolus species based on the 16S rRNA gene. A
connection stands for SparCC correlation with magnitude >0.9 (positive correlation–black edges) or < − 0.9 (negative correlation–red edges) and statistically
significant (P < 0.01). Each node represents taxa at OTU level and the size of node is proportional to the number of connections (degree). The color of the nodes is
based on the betweeness centrality, where darker colors indicate higher values. (For interpretation of the references to color in this figure legend, the reader is
referred to the web version of this article.)

structure of microbial community compared to P. filiformis, suggesting
that the rhizosphere of a wild plant species could present less capability
to recruit specific microbial groups. Since wild plant species can grow in
oligotrophic soils, it can corroborate the recruitment of similar micro­
bial groups in soil, such as k-strategist microbes (Ling et al., 2022).
4.2. Diversity and composition of the microbial communities
The rhizosphere of Phaseolus species recruits mainly Actinobacteria,
Proteobacteria, and Firmicutes, which present several bacterial groups
interacting in the rhizosphere of different legume plants, such as soy­
bean, cowpea, and lima bean (Albuquerque et al., 2022; Wang et al.,
2019), and acting as plant growth promoters (Boukhatem et al., 2022;
Felestrino et al., 2017). However, the rhizosphere can present microbial
groups that are detrimental to plants, competing for nutrients (Mohan­
ram and Kumar, 2019). However, we observed a significant variation in
phyla abundance comparing the rhizosphere of Phaseolus species. It
means that species belonging to the same genus recruit different mi­
crobial groups and reinforce the hypothesis of this study. The results
showed that Acidobacteria was more abundant in the rhizosphere of
P. lunatus, while Cyanobacteria was abundant in the rhizosphere of
P. microcarpus. Although Acidobacteria has been found to be abundant
in soils, a previous study has found Acidobacteria colonizing the
rhizosphere and promoting root growth in Arabidopsis (Kalam et al.,
2020). It could explain the observed correlation between root growth
and P. lunatus (Fig. 2B). In addition, a previous study has found Acid­
obacteria harboring genes encoding dinitrogenase (Ward et al., 2009),
being a promising bacterial group related to biological N fixation (BNF)
(Kielak et al., 2016). Therefore, this phylum could support BNF in
P. lunatus. Regarding Cyanobacteria, it is the first study reporting this
phylum in P. microcarpus. It is interesting since Cyanobacteria presents
the ability to colonize the rhizosphere (Lundberg et al., 2012) and
promote plant growth mainly through BNF (Franche et al., 2009).
Actinobacteria was found to be more abundant in the rhizosphere of
P. filiformis, P. acutifolius, and P. vulgaris. Although no information was
found about Actinobacteria in the rhizosphere of P. filiformis, and
P. acutifolius, previous studies have reported this phylum in the rhizo­
sphere of P. vulgaris (Mendes et al., 2018b; Pérez-Jaramillo et al., 2019).
The genetic differences between species from the Phaseolus genus
contributed to changing microbial diversity and richness, mainly con­
trasting the rhizosphere of P. lunatus and P. microcarpus. Distinct mi­
crobial diversity can be attributed to specific contrasts in C input in the
rhizosphere by the plant, where different plant species differ in the
quality and quantity of C resources they produce and release in the soil
(Berg and Smalla, 2009). Even different plant cultivars from the same
species influence the alpha diversity measurements (Liu et al., 2019;
Mendes et al., 2018b).
These differences observed in phyla composition, diversity, and
richness were also observed at a deeper taxonomical level. The analysis
showed an enrichment of specific microbial OTUs in each plant species.
In general, the rhizosphere community of P. lunatus and P. microcarpus
presented the highest number of enriched OTUs (clusters III and IV,
Fig. 4). These results suggest that each rhizosphere enriched specific and
exclusive microbial members, showing a species-specific response. Thus,
specific plant species, even those belonging to the same genus, recruits
exclusive rhizosphere colonizers (Bezemer et al., 2006). Interestingly,
the rhizospheric microbial community, at the genus level, differs be­
tween plant species, even if they belong to the same phylum (Aleklett

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A.C.A. Lopes et al.
Fig. 6. Boxplot showing the differential abundance of predicted functions related to N cycle among bulk soil and rhizosphere of different Phaseolus species based on
sequences of 16S rRNA gene. The prediction was made using the FAPROTAX database. Different lower-case letters refers to significant diffrences (P < 0.05) based on
post-hoc Tukey-Kramer test followed by Benjamini-Hochberg FDA correction.
et al., 2015). On the other hand, microbial OTUs grouped into clusters I,
II, and V were associated with the rhizosphere of P. filiformis, P. acuti­
folius, and P. vulgaris. Since these plant species are genetically close (>
85 % genetic similarity; Delgado-Salinas et al., 2006), mainly
P. acutifolius and P. vulgaris (99 % genetic similarity; Delgado-Salinas
et al., 2006) it contributed to this close similarity between microbial
communities in rhizospheres.
We then did a pairwise comparison between the microbial commu­
nity of each Phaseolus species with all others and the results showed
different microbial groups being enriched according to the Phaseolus
species. The rhizosphere of P. vulgaris enriched Rhizobiaceae, which
represent a diverse group of α-Proteobacteria collectively called rhizobia
that are involved in biological N fixation in P. vulgaris (Peralta et al.,
2016). Interestingly, Gaiellales was enriched in the rhizosphere of both
P. acutifolius and P. filiformis. Gaiellales belongs to the phylum Actino­
bacteria and has been found to be abundant in the rhizosphere (Zhou
et al., 2019), being aerobic and catalase and oxidase positive (Albu­
querque et al., 2011). In the rhizosphere, this bacterial family interacts
with other bacterial taxa, reducing nitrate and helping plants to assim­
ilate carbohydrates (Severino et al., 2019). The rhizosphere of P. lunatus
enriched Pyrinomonadaceae, while the rhizosphere of P. microcarpus
enriched Pirellulaceae. Bacteria belonging to Pyrinomonadaceae were
found in the rhizosphere of maize (Wang et al., 2021) and black pepper
(Umadevi et al., 2018), being known as a potential suppressor of path­
ogens in the rhizosphere. Thus, this bacterial group can assist P. lunatus
in suppressing pathogens in the rhizosphere.
4.3. Microbial co-occurrence network and N-related functional prediction
Network analysis was conducted to assess the complexity of the
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Applied Soil Ecology 182 (2023) 104731
interactions between microbial taxa in the rhizosphere of each Phaseolus
species. This analysis integrates and shows the connections of microbial
groups, and it has been applied in studies in the rhizosphere (Albu­
querque et al., 2022; Goss-Souza et al., 2019; Mendes et al., 2018b,
2018a; Pérez-Jaramillo et al., 2019). The results showed an increased
complexity of microbial interactions in both domesticated species
(P. vulgaris and P. lunatus) as compared to wild species (P. acutifolius, P.
microcarpus, and P. filiformis). It contrasts with previous studies which
showed the complexity of microbial connections in the rhizosphere
decreasing from wild to domesticated genotypes (Pérez-Jaramillo et al.,
2019; Rossmann et al., 2020; da Silva et al., 2022). However, a recent
study with maize has shown that the complexity of the microbial
network in the rhizosphere increased with domestication and even more
with plant breeding (Huang et al., 2022). Interestingly, it could explain
the highest complexity of microbial network in the rhizosphere of
P. vulgaris, which is considered a modern cultivar. According to Pérez-
Jaramillo et al. (2017), the root traits change among wild, domesticated,
and modern cultivars, so influencing microhabitats in the rhizosphere
and the co-occurrence network structure of the microbial communities.
Also, the increased complexity in the rhizosphere of P. vulgaris and
P. lunatus demonstrates that microbial communities from domesticated
species are more connected than microbial community assembly in the
rhizosphere of wild cultivars, which led us to suggest that the better
performance of domesticated plants depends on the strong interactions
of the rhizosphere microbiome with their environment and with the
plant.
The functional prediction aims to show the relationship between
microbial groups and their predictive potential functions, and it has
been applied in several studies with soil and rhizosphere (Albuquerque
et al., 2022; May et al., 2021; Merloti et al., 2019). The prediction of
A.C.A. Lopes et al.
potential functions associated to N cycle was compared among different
Phaseolus species and showed P. lunatus presented an increased abun­
dance of sequences linked to several steps of the N cycle. We focused on
the N cycle due to its importance to plant growth as a limiting nutrient in
terrestrial ecosystems (Henneron et al., 2020; Pedrinho et al., 2020). The
results evidenced that domestication affects both community composi­
tion and their functions in the rhizosphere. García-Palacios et al. (2013)
investigated how plant domestication influences litter quality and its
consequence on the nitrogen cycle, and they showed that soils where
domesticated plants were grown contained higher nitrate than soils with
wild cultivars. They argue that changes in plant traits during domesti­
cation have promoted higher litter quality, faster litter decomposition,
and, consequently, higher soil NO−3 availability. The results of this study
support this observation since the domesticated species P. lunatus pre­
sented the highest abundance of sequences affiliated to nitrite oxidation
(Fig. 6), which converts nitrite to nitrate. It is also worth noting that
Acidobacteria was more abundant in the rhizosphere of P. lunatus and
members of this phylum are involved in the nitrogen cycle. These results
show that the domestication process affects microbes related to the ni­
trogen cycle and suggest that the rhizosphere community from P. lunatus
is more efficient in processing nitrogen, which could be related to N
availability to the plant.
5. Conclusions
This study showed that changes in plant traits during domestication
have promoted changes in the rhizospheric microbial community of
plant species from the Phaseolus genus. Interestingly, these differences in
the microbial community associated with the rhizosphere are more
pronounced as greater the phylogenetic differences of the plant species.
The domesticated cultivars presented an enrichment of a higher number
of microbial OTUs, followed by increased network complexity and an
increased abundance of sequences affiliated to the N cycle. These data
revealed that the rhizospheric microbial community co-evolved with
plants during domestication and selected microbial groups with poten­
tial beneficial traits for the plant. However, considering that the soil is
the main source of microbial diversity for the rhizosphere (Banerjee and
van der Heijden, 2022), different soil types could shape the recruitment
of microbes in the rhizosphere. Thus, this study presents a limitation to
using only one type of soil, and further studies should evaluate these
plant species in different soil types and environmental conditions.
Nevertheless, the aim of our study was to evaluate the differences in the
assembly of the rhizosphere microbiome of different Phaseolus species
grown under similar conditions. Together, our results increase our
knowledge about how the domestication process affects the assembly of
the rhizospheric microbial communities and provide important infor­
mation that can drive future breeding programs.
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.apsoil.2022.104731.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data availability
Data will be made available on request.
Acknowledgments
Conselho Nacional de Desenvolvimento Científico e Tecnológico –
CNPq (305069/2018-1) and Coordenacao de Aperfeiçoamento de Pes­
soal de Nivel Superior – CAPES (Code 001). The DNA sequencing was
conducted at Centro de Genetica e Bioinformatica (CeGenBio) of
8
Applied Soil Ecology 182 (2023) 104731
Universidade Federal do Ceara (NPDM/UFC). Karla A. S. B. Brito, Jad­
son E.L. Antunes, Sandra M. B. Rocha, and Gerson N. Costa acknowledge
CAPES. Lucas W. Mendes, Angela C. A. Lopes, Vania M. M. Melo, Regina
L. F. Gomes, and Ademir S. F. Araujo acknowledge CNPq for their
fellowship of research.
Ethical approval
This article does not contain any studies with human participants or
animals performed by any of the authors.
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