Soil Biology and Biochemistry 189 (2024) 109292

Contents lists available at ScienceDirect

Soil Biology and Biochemistry

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

The interplay between Azospirillum brasilense and the native bacterial

communities in the soil and rhizosphere of maize (Zea mays L.)
Alexandre Pedrinho a, Lucas William Mendes b, Felipe Martins do Rêgo Barros a, João
William Bossolani c, Tayná Negri Kühn b, Maria Carolina Quecine d, Fernando Dini Andreote a, *
a
Department of Soil Science, “Luiz de Queiroz” College of Agriculture (ESALQ), University of São Paulo, Piracicaba, São Paulo, Brazil
b
Cell and Molecular Biology Laboratory, Center for Nuclear Energy in Agriculture (CENA), University of São Paulo, Piracicaba, São Paulo, Brazil
c
College of Agricultural Sciences, São Paulo State University (UNESP), Department of Crop Science, Botucatu, São Paulo, Brazil
d
Laboratory of Genetics of Microorganisms “Prof. Joao Lucio de Azevedo”, Department of Genetics, “Luiz de Queiroz” College of Agriculture (ESALQ), University of São
Paulo, Piracicaba, Brazil

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

Keywords: Azospirillum brasilense is a plant-growth promoting bacteria used as a bioinoculant in agriculture. However, the
Amplicon sequencing capacity of A. brasilense to establish itself in the soil and rhizosphere of different plants, as well as its interactions
Microbial ecology with the native soil bacterial community, are not fully understood. This knowledge gap can be attributed to
Microbial invasion
inconsistencies in the quantity of the inoculant delivered (population size) and abiotic and biotic factors that
Microbial interaction
Plant growth-promoting bacteria (PGPB)
modulate its performance. In this study, our objective was to gain a better understanding of how different-sized
populations (sub-dose, recommended dose, and super-dose) of A. brasilense affect and interact with the structure,
diversity, and connections of the native bacterial community in both bulk soil and rhizosphere of maize plants at
different growth stages. Furthermore, we aimed to assess the effectiveness of these different-sized populations of
A. brasilense in promoting plant growth. The introduction of a large population (recommended dose and super-
dose) of A. brasilense significantly improved maize growth parameters, while a small population (sub-dose) did
not. In the bulk soil, we observed that a large population of A. brasilense was capable of initially disrupt the native
soil bacterial community. However, the native soil bacterial community was able to recover from the momentary
disturbance and return to its initial state. In the rhizosphere of maize plants, large populations of A. brasilense
extended their impact on the native bacterial community, possibly due to permanent changes in plant traits, such
as root morphology and exudation. Furthermore, the co-occurrence network analysis revealed shifts in keystone
taxa (i.e., taxa that confer high connectivity) in the rhizosphere of maize plants. A. brasilense played a key role at
the early stages, but it was later replaced by Rhizobiales, a native soil bacterium. This study presents novel
evidence of how different-sized populations of A. brasilense can influence microbe-microbe and plant-microbe
interactions, ultimately affecting maize growth.

Microbial inoculants are gaining acceptance and prominence in
agriculture (Waltz, 2017). Among the microorganisms globally utilized
in agriculture, Azospirillum brasilense stands out as one of the most
attractive alternatives to improve crop performance and reduce the
dependency on synthetic nitrogen fertilizers (Hungria et al., 2022). This
is because A. brasilense, a free-living microorganism, has the ability to
perform biological nitrogen fixation (Day and Döbereiner, 1976) and
produce several phytohormones, including auxins, cytokinins, ethylene,
and gibberellins (Cassán and Diaz-Zorita, 2016). Additionally, it can
establish beneficial associations with plants, modifying root architecture
(Cassán et al., 2020), enhancing nutrient and water uptake, and often
resulting in increased plant growth and yields (Hungria et al., 2022).
However, the inconsistent efficacy of microbial inoculants (including
A. brasilense) prevents their widespread adoption as a standard agri­
cultural practice worldwide (Mitter et al., 2021). The variability in the
efficacy of microbial inoculants, especially in field conditions, may be
attributed to the quantity of the inoculant delivered (population size)
and abiotic (soil pH and nutrient content) and biotic (competition and
predation) factors that can hinder the establishment and survival of the
introduced microbe (French et al., 2021).

* Corresponding author.
E-mail address: fdandreo@usp.br (F.D. Andreote).

https://doi.org/10.1016/j.soilbio.2023.109292
Received 1 October 2023; Received in revised form 13 December 2023; Accepted 17 December 2023
Available online 21 December 2023
0038-0717/© 2023 Elsevier Ltd. All rights reserved.
A. Pedrinho et al. Soil Biology and Biochemistry 189 (2024) 109292

To assess how different-sized populations of A. brasilense affect maize
performance and the native soil bacterial community in both bulk soil
and rhizosphere of maize, we performed a pot experiment under control
conditions. We hypothesized that (i) the introduction of a large popu­
lation of A. brasilense may initially disrupt the structure, diversity, and
connections of the native soil bacterial community, consequently
affecting maize performance. However, we predict that (ii) the native
soil bacterial community will exhibit resilience and eventually recover
from this initial disturbance. Maize seeds were sorted into three distinct
groups and inoculated with a sub-dose (0.1 × the recommended dose),
recommended dose (1.0 × - consists of 2 × 1010 CFUs per 60,000 seeds),
and super-dose (10.0 × the recommended dose) of A. brasilense Ab-V5.
Additionally, a control group was included (ddH2O). Maize plants and
soil samples, including bulk soil and the rhizosphere, were collected at
both 15 and 30 days after the inoculation (DAI). The dry biomass of
maize shoots and roots was assessed. Total DNA was extracted and
submitted for bacterial 16S rRNA amplicon sequencing. The bacterial
community structure was assessed by principal component analysis
(PCA) along with permutational multivariate analysis of variance
(PERMANOVA; Anderson, 2001). The observed OTUs (Richness) and
Shannon diversity index were also calculated. Lastly, network analysis
was performed using the Python module “SparCC” (Friedman and Alm,
2012) and graphs were created using the Gephi software (Bastian et al.,
2009). A full description of experiment design and analysis is provided
in the Supplementary Material.
A. brasilense had a positive effect on the growth of maize plants. We
observed that the recommended dose and super-dose of A. brasilense
inoculant generally increased the shoot, root, and total plant dry mass of
maize when compared to the sub-dose and control at both sampling
periods (P < 0.05; Fig. 1A–F). In this context, we argue that the large
population of A. brasilense introduced had a better chance of establishing
an initial plant-microbial association with maize (Bashan and
de-Bashan, 2010), promoting significant changes in the root system (e.g.,
root morphology and physiology), and consequently enhancing plant
growth (Cassán et al., 2020).
When specifically looking at the potential impacts of A. brasilense on
the soil bacterial community structure, we observed that in the bulk soil,
samples receiving the recommended dose and super-dose clustered
together, separate from the control at 15 DAI (Fig. 1G). Additionally,
these two doses increased both bacterial richness and diversity when
compared to the control (P < 0.05; Fig. 2A, C). The initial impact of
these doses can be associated to the intensive competition between the
large population of A. brasilense introduced and the native soil bacterial
community for both ecological niches (Eisenhauer et al., 2013) and
nutrient resources (Yang et al., 2017). In this competition, we observed
that the native soil bacterial community, characterized by its high di­
versity and adaptability to the local environment, was able to overcome
the temporary disturbance imposed by the large population of
A. brasilense introduced and return to its initial state after 30 DAI
(Figs. 1H and 2A, C; Liu et al., 2022).
In the rhizosphere, regardless of the dose used, no significant
changes in the bacterial community structure, richness, and diversity
were observed with the application of the inoculant at 15 DAI (P > 0.05;
Figs. 1I and 2B, D). Conversely, at 30 DAI, the bacterial community

Fig. 1. Dry shoot (A-B), root (C-D), and total biomass (E-F) of maize plants at both 15 and 30 days after the inoculation (DAI) with Azospirillum brasilense. The
inoculation included a range of doses: (i) sub-dose (0.1 × ), (ii) recommended dose (1.0 × ), and (iii) super-dose (10.0 × ) of the microbial inoculant. Bars labeled
with different lowercase letters indicate significant differences among the treatments at 15 DAI, as determined by Tukey’s post hoc test (P < 0.05). Bars labeled with
different uppercase letters indicate significant differences among the treatments at 30 DAI, also determined by Tukey’s post hoc test (P < 0.05). Error bars represent
the standard error of four independent replicates. Principal component analysis (PCA) based on bacterial 16S rRNA from both bulk soil and rhizosphere of maize
plants at 15 and 30 days after the inoculation (DAI) with A. brasilense. The inoculation included a range of doses: (i) sub-dose (0.1 × ), (ii) recommended dose (1.0 ×
), and (iii) super-dose (10.0 × ) of the microbial inoculant. (G-H) The structure of the bacterial communities in the bulk soil at 15 and 30 days after the inoculation
(DAI) with A. brasilense; and (I-J) The structure of the bacterial communities in the rhizosphere of maize plants at 15 and 30 days after the inoculation (DAI) with A.
brasilense. Analysis of permutation (PERMANOVA) based on the Bray-Curtis similarity index is indicated in the upper right corner of the graph.

2
A. Pedrinho et al. Soil Biology and Biochemistry 189 (2024) 109292

Fig. 2. Richness and diversity measurements of bacterial communities in both bulk soil and rhizosphere of maize plants at 15 and 30 days after the inoculation (DAI)
with Azospirillum brasilense. The inoculation included a range of doses: (i) sub-dose (0.1 × ), (ii) recommended dose (1.0 × ), and (iii) super-dose (10.0 × ) of the
microbial inoculant. (A–B) Richness and (C–D) diversity measurements were assessed based on bacterial 16S rRNA at the OTU level, with a similarity threshold of
97%. Box plot labeled with different lowercase letters indicate significant differences among the treatments at 15 DAI, as determined by Tukey’s post hoc test (P <
0.05). Box plots labeled with different uppercase letters indicate significant differences among the treatments at 30 DAI, also determined by Tukey’s post hoc test (P
< 0.05). Network co-occurrence analysis of bacterial communities based on the 16S rRNA gene in the (E–F) bulk soil and (G–H) rhizosphere of maize plants at 15 and
30 days after the inoculation (DAI) with A. brasilense. The inoculation included a range of doses: (i) sub-dose (0.1 × ), (ii) recommended dose (1.0 × ), and (iii) super-
dose (10.0 × ) of the microbial inoculant. A connection stands for SparCC correlation with magnitude >0.9 (positive correlation - blue edges) or < − 0.9 (negative
correlation - red edges) and statistically significant (P < 0.01). Each node represents taxa at the OTU level, and the size of the node is proportional to the number of
connections (degree). The color of the nodes is based on the betweenness centrality, where darker colors indicate higher values.

structure was significantly affected by the A. brasilense inoculation, with
samples receiving the recommended dose and super-dose clustering
together, separate from the sub-dose and control (Fig. 1J). This late
impact on the bacterial community structure in the rhizosphere of maize
can be attributed to permanent changes in plant traits (e.g., root
morphology and physiology – supported by Fig. 1A–F) (Kong and Liu,
2022; Zuluaga et al., 2021), which may have affected key bacterial
groups involved in different processes, including nutrient cycling, plant
growth/defense, etc (Rocha et al., 2023).
We then used the co-occurrence network analysis to assess the po­
tential impacts of A. brasilense inoculation on the dynamics of the native
bulk soil and rhizosphere bacterial communities. In the bulk soil, the
analysis indicated that the complexity of connections among microor­
ganisms increased with the application of the super-dose compared to
the control (Fig. 2E and F; Supplementary Table ST3). Conversely, in the
rhizosphere of maize, we observed the opposite effect, with the super-
dose reducing the complexity when compared to the control (Fig. 2G
and H; Supplementary Table ST4). According to Mendes et al. (2017),
biological disturbances (e.g., changes in soil properties, fertilization,
microbial inoculation, etc.) can lead to a temporary and/or permanent
increase in taxonomic and functional diversity, consequently reflecting
in a more complex ecological network.
Lastly, we tackled bacterial keystones in the co-occurrence networks
(Supplementary Table ST5-8). According to Banerjee et al. (2018),
keystones play important biological and ecological roles in the micro­
biome. They have a large influence in the microbial community,
conferring high connectivity and their removal can cause significant
changes in microbial community composition and functioning. In the
bulk soil, the family Burkholderiaceae was identified as one of the
important keystones in the control, while the genera Flavisolibacter and
Flaviaesturariibacter played crucial roles in the treatment that received
the super-dose of the inoculant. Both Flavisolibacter and
Flaviaesturariibacter possess a versatile metabolism, are resistant to stress
conditions, and are involved in nutrient cycling (Battistuzzi and Hedges,
2009; Tebele et al., 2023). Therefore, it is highly possible that the
replacement and/or removal of bacterial keystones after A. brasilense
inoculation led to significant changes in the microbial-microbial in­
teractions, setting the stage for other keystones to dominate in an altered
microbial community (Banerjee et al., 2018; Solé and Montoya, 2001).
In the rhizosphere at 15 DAI, Caulobacteraceae and Edaphobacter
were identified as important keystones in the control, while A. brasilense
played a crucial role in the treatment that received the super-dose of the
inoculant. This finding supports our initial idea/findings that the large
population of A. brasilense introduced had a better chance of establishing
an early plant-microbial association with maize (Bashan and de-Bashan,
2010). However, our results also demonstrate that A. brasilense was not
able to withstand the abiotic and biotic pressures for a long period.
A. brasilense was either (i) outcompeted and “killed” by the native soil
bacterial community and/or (ii) most likely survived but with reduced
relative abundances (Bashan et al., 1995; Bauer et al., 2018; Supple­
mentary Figure SF1), enabling the native soil microbiome to rearrange
its structure and composition to attenuate its impact (Mallon et al.,
2018). This is evident at 30 DAI, where Rhizobiales, another PGPB
capable of performing nitrogen fixation, producing different phytohor­
mones, and protecting plants against environmental stress (Erlacher
et al., 2015; Garrido-Oter et al., 2018), was identified as a new keystone
taxa. In this context, we emphasize that the loss and/or replacement of a
keystone taxa can have a profound impact on ecosystem interactions and
services if their functions are not replaced by other bacterial taxa with
the same features (i.e., functional redundancy) and/or replaced by less
efficient ones (Chapin et al., 2000).
Here, we described for the first time how different-sized populations
of A. brasilense affect maize performance and the structure, diversity,
and connections of the native soil bacterial community in both bulk soil

3
A. Pedrinho et al.
and its rhizosphere. Our findings highlight the dynamic nature of mi­
crobial interactions, in both the bulk soil and rhizosphere, where
microbe-microbe and plant-microbe interactions play crucial roles in
shaping microbial communities and ecosystem services. Understanding
these dynamics is essential for harnessing the potential of PGPB (such as
A. brasilense) in sustainable agriculture and soil management practices,
while also considering the ecological consequences of their introduction
and persistence in soil ecosystems.
CRediT authorship contribution statement
Alexandre Pedrinho: Writing – review & editing, Writing – original
draft, Visualization, Validation, Software, Resources, Methodology,
Investigation, Funding acquisition, Formal analysis, Data curation,
Conceptualization. Lucas William Mendes: Writing – review & editing,
Writing – original draft, Visualization, Validation, Supervision, Soft­
ware, Methodology, Investigation, Formal analysis, Conceptualization.
Felipe Martins do Rêgo Barros: Writing – review & editing, Writing –
original draft, Visualization, Validation, Investigation, Formal analysis,
Conceptualization. João William Bossolani: Writing – review & edit­
ing, Writing – original draft, Investigation, Formal analysis, Data cura­
tion. Tayná Negri Kühn: Writing – original draft, Investigation, Formal
analysis. Maria Carolina Quecine: Writing – review & editing, Writing
– original draft, Resources, Methodology. Fernando Dini Andreote:
Writing – review & editing, Writing – original draft, Visualization,
Validation, Supervision, Resources, Project administration, Methodol­
ogy, Investigation, Funding acquisition, Formal analysis,
Conceptualization.
Declaration of competing interest
The authors declare the following financial interests/personal re­
lationships which may be considered as potential competing interests:
Alexandre Pedrinho reports financial support was provided by The Sao
Paulo Research Foundation (FAPESP). Fernando Dini Andreote and
Lucas William Mendes report financial supports were provided by Na­
tional Council for Scientific and Technological Development (CNPq).
Data availability
Data will be made available on request.
Acknowledgments
This study was supported by a grant from Fundação de Amparo à
Pesquisa do Estado de São Paulo (FAPESP 2019/16006-4). We also
thank Conselho Nacional de Desenvolvimento Científico e Tecnológico
(CNPq) for grant 308611/2021-7 for Fernando D Andreote and 307670/
2021-0 for Lucas W Mendes.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.soilbio.2023.109292.
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