Applied Soil Ecology 170 (2022) 104297

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Applied Soil Ecology

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

Effects of inoculation with plant growth-promoting rhizobacteria from the

Brazilian Amazon on the bacterial community associated with maize
in field
Jessica Aparecida Ferrarezi a, 1, Paula de Almeida Carvalho-Estrada a, 1, Bruna Durante Batista b, 1,
Rafael Martins Aniceto a, Bruno Augusto Prohmann Tschoeke a,
Pedro Avelino de Maia Andrade a, Bruna de Moura Lopes a, Maria Leticia Bonatelli a,
Estácio Jussie Odisi c, João Lucio Azevedo a, Maria Carolina Quecine a, *
a
Department of Genetics, University of São Paulo, College of Agriculture “Luiz de Queiroz”, Piracicaba, SP 13418-900, Brazil
b
Hawkesbury Institute for the Environment, Western Sydney University, Richmond, NSW 2753, Australia
c
Biome4All, São Paulo, SP 01419-909, Brazil

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

Keywords: The objective of this study was to investigate the effects of the inoculation with the plant growth-promoting
Zea mays rhizobacteria Bacillus thuringiensis RZ2MS9 and Burkholderia ambifaria RZ2MS16, both from the Brazilian
Inoculant Amazon, on the bacterial community of the rhizobiome and leaves of maize grown in field. For comparison, we
PGPB
analysed the effects of inoculating Azospirillum brasilense Ab-V5, a strain that is commercialized as inoculant for
Azospirillum brasilense
maize, as well as the combinations RZ2MS16 + Ab-V5 and RZ2MS9 + Ab-V5. The treatment RZ2MS9 + Ab-V5
Bacillus thuringiensis
Burkholderia yielded the highest plant height and stalk diameter, which were significantly different from the non-inoculated
control (p < 0.05). The core microbiome of maize was mainly composed of the bacterial classes Gammapro­
teobacteria, Betaproteobacteria, Actinobacteria, Alphaproteobacteria, Cytophagia, and Bacilli. Overall, the
inoculation process had no effect either on the composition of the maize-associated bacterial community or on
the total bacterial biomass. However, we detected significant differences in the richness and in the community
structure among the plant niches analysed. Linear discriminant analysis identified that the class Actinobacteria,
and the order Actinomycetales were enriched in leaf and root of plants treated with RZ2MS16 + Ab-V5 and in
rhizosphere of plants treated with RZ2MS9 + Ab-V5. Functional analysis of the soil samples revealed significant
differences in the abundance of predicted genes encoding proteins related to respiration and solubilization of Cu,
Mg, and K among treatments. Furthermore, the NMDS ordination showed association between different func­
tional gene categories and some plant traits. Our findings contribute to understanding the efficacy of microbial
inoculants for maize in field conditions.

1. Introduction
The contribution of agriculture to the global economy has been
growing in recent decades, increasing the use of agrochemicals and their
negative impacts on the environment and human health (FAO, 2020).
For instance, the accumulation of synthetic pesticides in the soil can
cause deterioration of soil properties and reduce plant growth or make
the plant unsuitable for consumption (Pascale et al., 2020). At the same
time, the production of primary crops such as maize needs to increase
significantly in the coming decades to meet the needs a growing world
population (FAO, 2020). Thus, a production system based on sustainable
agriculture is urgently required especially for primary crops.
One emerging strategy to promote sustainable agriculture is the use
of inoculants formulated with Plant Growth-Promoting Bacteria (PGPB)
(Aydinoglu et al., 2020; Florio et al., 2019; Hu et al., 2017; Ullah et al.,
2020). PGPB can benefit plant growth directly by facilitating the uptake
of soil nutrients when employing biological nitrogen fixation or miner­
alization/solubilization of phosphate. They can also provide

* Corresponding author.
E-mail address: mquecine@usp.br (M.C. Quecine).
1
These authors have contributed equally to this work and share first authorship.

https://doi.org/10.1016/j.apsoil.2021.104297
Received 10 June 2021; Received in revised form 21 August 2021; Accepted 27 October 2021
Available online 8 November 2021
0929-1393/© 2021 Elsevier B.V. All rights reserved.
J.A. Ferrarezi et al.
phytohormones to plants and protect them against biotic and abiotic
stresses. Indirect benefits include priming the plant's resistance response
against stresses (Trivedi et al., 2020).
Although inoculants containing PGPB have been applied to plant
crops for over 120 years (Arora et al., 2017), the inconsistent efficacy of
these products, especially in field conditions, prevent the adoption of
microbial inoculants as a standard agricultural practice worldwide
(Mitter et al., 2021; Naamala and Smith, 2020). The inconsistent effi­
cacy of microbial inoculants in field may be explained by several un­
controlled biotic and abiotic factors that can work against the
introduced microbe's survival (French et al., 2021). In addition, the in­
oculants can be incompatible with other products applied to crops or can
even be defeated by the indigenous plant-associated microbiota per­
sisting in low and ineffective levels in the soil (French et al., 2021). Thus,
testing microbial products in field conditions and analysing their
compatibility with other products and their effects on the indigenous
plant microbiota is crucial for understanding the persistence and effi­
cacy of such products.
Recent advances in high-throughput sequencing have allowed re­
searchers to portray the plant-associated microbiome community as­
sembly under different conditions as well as the microbial recruitment to
host tissues over space and time (Dickey et al., 2020). These tools will
greatly assist in the development of bioinoculants formulated with PGPB
that work effectively in a complex agricultural setting, such as the field,
for improved and sustainable plant production.
The Brazilian Amazon is a biodiversity hotspot containing unique
plant species such as the Amazonian guarana (Paullinia cupana). Studies
about the microbes associated with these plants are scarce (Azevedo
et al., 2000; Batista et al., 2018; Bogas et al., 2015; Bonatelli et al., 2016,
2019). Therefore, it is possible that the Amazon is a valuable source of
unknown microbes with unexplored biotechnological potential that can
be used as inoculants in agriculture. The PGPB Burkholderia ambifaria
strain RZ2MS16 (Batista et al., 2016b) and Bacillus thuringiensis strain
RZ2MS9 (Batista et al., 2016a, 2021) were previously isolated from the
guarana rhizosphere by our group (Batista et al., 2018). These strains
remarkably promoted the growth of maize in greenhouse conditions.
Increases of around 250% and 140% in maize root dry weight were
obtained by applying RZ2MS9 and RZ2MS16, respectively, as compared
to the non-inoculated control, 60 days after seeding (Batista et al.,
2018). However, the effects of these strains on maize growth and pro­
ductivity under field conditions have not been evaluated until now.
The commercial use of the bacterium Azospirillum brasilense strain
Ab-V5 on maize has been growing exponentially in Brazil (Fukami et al.,
2016, 2017; Hungria et al., 2010; Marks et al., 2015). The first inoculant
containing A. brasilense produced in the country was released to the
market in 2009 and, ten years later, it is estimated that about 10.5
million doses of inoculants carrying Ab-V5 were applied in cereal crops
in Brazil (Santos et al., 2021). Considering the high adoption of
A. brasilense products by Brazilian farmers, it is desirable that any new
microbial product for maize be compatible with these strains. Several
studies suggest that a consortium of microbes can be more effective in
increasing disease resistance and promoting plant growth than the
inoculation of single bacterial strains (Armanhi et al., 2018; Hungria
et al., 2010, 2015, 2016; Lopes et al., 2021). Therefore, the present study
analysed the effect of B. ambifaria RZ2MS16, B. thuringiensis RZ2MS9,
and A. brasilense Ab-V5, as single strains and as consortia on the growth
of maize and on the assembly of maize-associated bacterial communities
under field conditions.
2. Materials and methods
2.1. Bacterial strains and plant material
Three PGPB strains were used in this study: Burkholderia ambifaria
RZ2MS16, Bacillus thuringiensis RZ2MS9, and Azospirillum brasilense Ab-
V5. The first two strains belong to the Laboratory of Genetics of
2
Applied Soil Ecology 170 (2022) 104297
Microorganisms “Prof. João Lucio de Azevedo” at the University of São
Paulo. Both strains have been deposited at the Brazilian Collection of
Environmental and Industrial Microorganisms (CBMAI). RZ2MS16 and
RZ2MS9 are routinely grown in Luria-Bertani (LB) medium (Sambrook
and Russell, 2001) at 28 ◦ C.
The A. brasilense strain Ab-V5 has been kindly provided by Dr.
Mariangela Hungria from the Culture Collection of Diazotrophic and
Plant Growth Promoting Bacteria of “Embrapa Soja”. The strain is
routinely grown in Dextrose Yeast Glucose Sucrose (DYGS) medium
(Rodrigues Neto et al., 1986) at 28 ◦ C. Conventional maize (Zea mays L.)
seeds, semi-flint hybrid DKB 390 (Dekalb, Monsanto), were used in the
field trial.
2.2. In vitro compatibility of PGPB strains
The compatibility among RZ2MS16, RZ2MS9, and Ab-V5 was veri­
fied in pairwise combinations using the plate confrontation method
described by Loaces et al. (2011) with modifications. Briefly, each strain
was inoculated into its respective liquid medium and incubated at 28 ◦ C
and 180 rpm for 8 h. Bacterial suspensions (each strain containing 1 ×
108 CFU⋅ml− 1) were spread on plates containing their respective agar
medium, which were allowed to air-dry in a laminar-flow cabinet. Then,
four drops (10 μl each) of the other bacterial suspension (containing 1 ×
108 CFU⋅ml− 1) were inoculated on the same plate. The experiment was
performed in three replicates, and we tested all possible combinations
among the three tested strains. After the inoculation, the plates were
incubated at 28 ◦ C for 24 h. A growth inhibition zone around the bac­
terial colonies indicated incompatibility between the two strains tested.
2.3. Field experiment set-up
Bacterial pre-inoculums were prepared by growing each bacterial
strain at 28 ◦ C and 150 rpm, in its respective culture medium, for 24 h.
Initial cell density was adjusted to 1 × 108 CFU⋅ml− 1 in 50 ml of liquid
medium. Then, the bacterial suspensions were grown at 28 ◦ C and 150
rpm for 8 h and adjusted to a final cell density of 1 × 109 CFU⋅ml− 1 for
each bacterial inoculum. Cell density was determined using the LB
medium microdrop technique (Estrada-Bonilla et al., 2017, 2021). The
inoculums were shortly kept at room temperature prior to seed
inoculation.
For inoculation, the maize seeds were washed with sterile distilled
water and rinsed in a sterile 10% (w/v) sucrose solution for 10 min.
Then, the seeds were drained and dried at room temperature. After
drying, they were submerged into the bacterial treatments and incu­
bated by shaking at 45 rpm for 30 min at room temperature. The control
treatment involved washing the seeds and treating them with the su­
crose solution as previously described and submerging them into a 1:1
mix of the LB and DYGS media with no bacterial growth.
The seeds were manually sowed with the aid of a hoe in a field
located in the “Luiz de Queiroz” College of Agriculture in Piracicaba-SP,
Brazil (22◦ 42′ 23′′ S, 47◦ 38′ 14′′ W, 535 m altitude, Köppen-Geiger climate
type Cfa). The soil in the area is a Dystrophic Red Latosol (Rhodic
Hapludox) (Soil Survey Staff, 2010). Soil fertility was corrected by
adding 290 kg ha− 1 of 8-28-16 NPK fertilizer in the whole experimental
area.
The experiment was conducted in a randomized complete blocks
design with five blocks (replicates). The six treatments were: (i) single
inoculation of B. thuringiensis RZ2MS9 (RZ2MS9 hereafter); (ii) single
inoculation of B. ambifaria RZ2MS16 (RZ2MS16 hereafter); (iii) single
inoculation of A. brasilense Ab-V5 (Ab-V5 hereafter); (iv) co-inoculation
of RZ2MS9 and Ab-V5 (RZ2MS9 + Ab-V5 hereafter); (v) co-inoculation
of RZ2MS16 and Ab-V5 (RZ2MS16 + Ab-V5 hereafter); and (vi) non-
inoculated control. Maize seeds were sown in the 5 m × 8 m blocks,
which were composed of 11 rows that were 80 cm distant from each
other. Each row in a block corresponded to a plot. From the 11 plots per
block, 6 plots received the treated seeds, and 5 plots received seeds with
J.A. Ferrarezi et al.
no treatment, these plots were used as buffers between the treatments
plots to avoid inoculants mixing in the soil by runoff. The distance be­
tween the blocks was of 1 m (Supplementary Fig. 1). Twenty seeds were
sown per plot.
2.4. Assessment of the treatments' effect on the maize growth and yield
Sixty days after sowing (DAS), at the R1 growth stage (silking stage),
we collected leaf (5 cm2 of the middle third of the second leaf below the
cob), root, and rhizosphere soil samples from one randomly selected
plant in each plot (5 plants per treatment). The samples were ice-cold
transported to the laboratory, where they were kept at 4 ◦ C until
being processed for DNA extraction. On the same day, we counted the
number of plants per plot, and measured the plant height, cob insertion
height (ear height), and stalk diameter of all plants in the experiment.
The stalk diameter was measured using a digital pachymeter at the first
knot under the cob. At the end of the growth cycle (after physiological
maturity), the whole experiment was harvested, and the cobs were
classified by plot and counted. The cob length was measured, and the
grains of all cobs were mechanically detached and weighed. One hun­
dred grains per plot were randomly selected and weighed. The dry
matter of grains was measured, and the grains' moisture was used to
correct the estimate yield per hectare for each treatment.
2.5. DNA extraction and quantification of bacterial communities
DNA extractions from leaves, roots, and rhizosphere soil samples
were performed using DNeasy® Plant Mini Kit (Qiagen), following the
manufacturer's instructions. The integrity of the extracted DNA was
determined by 1.0% (w/v) agarose gel electrophoresis with SYBR® Safe
(Life Technologies). The concentration of DNA was estimated by spec­
trophotometer NanoDrop™ (NanoDrop Technologies).
The number of 16S rRNA gene copies was estimated per nanogram of
total DNA by quantitative PCR using a IQ™5 (Bio-Rad) Real-Time PCR
Detection System with SYBR® Green I. We used the primers 534F (5′ –
CCA GCA GCC GCG GTA AT –3′ ) and 783R (5′ – ACC MGG GTA TCT AAT
CCK G –3′ ) (Rastogi et al., 2010) to quantify the bacterial community.
Amplification was performed by applying the following conditions:
95 ◦ C for 3 min, 40 cycles at 95 ◦ C for 30 s, 53 ◦ C for 30 s, and 72 ◦ C for
30 s. The final reaction volume was 25 μl, which contained 12.5 μl of
Platinum® Quantitative PCR SuperMix-UDG (Invitrogen), 0.20 μM of
each primer, 5 ng of template DNA and Milli-Q water to complete the
reaction volume. Standard curves were obtained by performing ampli­
fications with the number of known copies of the template DNA added to
the reaction with the primers POF27F (5′ – GAG AGT TTG ATC CTG GCT
GAC –3′ ) and 1378R (5′ – CGG TGT GTA CAA GGC CCG GGA ACG − 3′ )
(Heuer et al., 1997) using E. coli DNA. The amplification data of DNA
extracted from the samples were interpolated with the standard curve to
determine the number of copies of the gene of interest (Estrada-Bonilla
et al., 2017).
2.6. 16S rRNA gene sequencing
The extracted DNA was amplified using the primers 799F (5′ – AAC
MGG ATT AGA TAC CCK G –3′ ) and 1492R (5′ – TAC GGY TAC CTT GTT
ACG –3′ ) (Chelius and Triplett, 2001) to avoid amplification of chloro­
plast DNA. The amplification conditions were determined for a final
reaction volume of 50 μl, composed of 1× PCR Buffer, 2.5 mM MgCl2,
0.2 μM dNTP, 0.2 μM of each primer, 0.4 mM BSA, 2 U of Platinum® Taq
DNA Polymerase (Invitrogen), 50 ng of template DNA and Milli-Q water
to complete the reaction volume. PCR reactions and band excision
procedures were carried out according to Carvalho-Estrada et al. (2020).
PCR products were purified in 1% (w/v) agarose gel, with GFX™
PCR DNA and Gel Band Purification Kit (GE Healthcare), following the
manufacturer's instructions. After purification, these samples were used
as the DNA template in a new reaction using the primer set 967F (5′ –
3
Applied Soil Ecology 170 (2022) 104297
CAA CGC GAA GAA CCT TAC C –3′ ) and 1046R (5′ – CGA CAG CCA TGC
ANC ACC T –3′ ) for the V6 region of 16S rRNA gene (Sogin et al., 2006).
Barcodes containing a set of five nucleotides were added to the primer
967F, which were used as markers for each sample. The amplification
conditions were as previously described.
All fragments containing barcodes were mixed at equimolar con­
centrations and purified with the ChargeSwitch® PCR Clean-Up Kit
(Invitrogen) and quantified with Qubit® 2.0 Fluorometer (Life Tech­
nologies). Subsequently, samples were prepared to be loaded on a 316
chip for posterior sequencing (Ion Sequencing Kit User Guide v2.0, Life
Technologies) using the Ion Personal Genome Machine™ (PGM™) (Ion
Torrent, Life Technologies). The procedures for library preparation were
carried out at the Environmental Microbiology Laboratory, at “Embrapa
Meio Ambiente” (São Paulo, Brazil).
2.7. Sequencing data analysis
Sequencing data were processed with tools from USEARCH pipeline
v.11 (Edgar, 2010) and Ribosomal Database Project (RDP) (Cole et al.,
2014). The raw reads were pre-processed for quality control, with
adapters trimmed and data filtered. The main criteria for trimming were
sequencing oligonucleotides and barcodes length, and low-quality bases
at 3′ extremes (quality score < 20). For filtering, the threshold of
maximum error rate of 3% was established. After that, the reads were
oriented to the RDP database to improve data annotation, submitted to
dereplication (removal of redundant reads) and to singletons removal
(single reads or low-quality ones), and finally clustered at 100% identity
to form ZOTUs (zero-radius OTU). The ZOTUs were counted per sample
and annotated with RDP taxonomy information. At the end of the
analysis, four files were generated and submitted to analysis at Micro­
biomeAnalyst platform (www.microbiomeanalyst.ca): The ZOTUs count
table (which records the number of times each ZOTU was observed in
each sample), the metadata sheet, the ZOTUs mapping file, and the
Newick phylogenetic tree. Phylogenetic distances between ZOTUs were
estimated and used for the creation of Newick phylogenetic trees (Junier
and Zdobnov, 2010). The sequence data reported in this study are
available at the MG-RAST repository (Meyer et al., 2008) under the IDs
487318 (root and leaf samples) and 487340 (rhizosphere soil samples).
To obtain the functional profile of the soil samples, their read data
were reprocessed with DADA2 using QIIME2 v. 2020.8 (Bolyen et al.,
2019) and PICRUSt2 (Douglas et al., 2020) for the functional prediction
(full pipeline, excluding sequences with NTSI > 2), following the Agri-
Analysis pipeline established by Biome4All. A database of maize crop
soil was provided by Biome4All (São Paulo, Brazil) for comparison. The
database is organized into curated sets of functional categories derived
from MetaCyc and KEGG pathways. The distribution of percentiles
across samples for each set of pathways was used for the direct com­
parison. For each functional category, a score was assigned that was
equivalent to its placement in relation to the percentiles, according to
Biome4All's pipeline. The set of scores for the samples were assessed
with non-metric multidimensional scaling (NMDS), using the metaMDS
function for the VEGAN R package with the Bray-Curtis distance matrix
(Dixon, 2003). Envfit (999 permutations) was used to fit the vegetable
growth parameters into the ordination.
2.8. Statistical analyses
Plant growth data were analysed by a one-way analysis of variance
(ANOVA) using R software v. 3.5.2 (R Core Team). A post hoc test
(Fisher's Least Significant Difference – LSD) was performed to determine
pairwise differences among the means of each phenotypic attribute at
5% of significance. The log10 number of 16S rRNA gene copies was
analysed by LSD test at 5% significant level using the R software.
The changes in the bacterial community diversity and composition
were assessed by analyses of alpha- and beta-diversity. The alpha-
diversity estimates were performed using the online tool
J.A. Ferrarezi et al. Applied Soil Ecology 170 (2022) 104297

MicrobiomeAnalyst (Chong et al., 2020; Dhariwal et al., 2017). We 3. Results

generated the indices Chao 1 (richness) and Shannon (richness and
evenness) using the online platform and analysed the differences be­
tween sample groups using a t-test/ANOVA in R. Principal coordinates
analysis (PCoA) based on the Bray-Curtis index distance method (Bray
and Curtis, 1957) was plotted using the online platform of Micro­
biomeAnalyst. We used PERMANOVA to evaluate the differences in the
bacterial community structure among treatments.
To establish the correlation between the bacterial community
structure and the phenotypic variables, a principal component analysis
(PCA) with a biplot was generated using R software (R Core Team).
Linear discriminant analysis (LDA) coupled with effect size (LEfSe;
http://huttenhower.sph.harvard.edu/galaxy/root) was used to identify
the bacterial taxa differentially represented among the treatments. The
criterion for LEfSe was set as logarithmic LDA > 2.0 and the Wilcoxon p-
value < 0.05.
3.1. Compatibility assay among bacterial strains
The pairwise compatibility assay showed no negative interactions
among the strains used in this study. In other words, the strain Ab-V5
does not inhibit the growth of either RZ2MS9 or RZ2MS16 (Supple­
mentary Fig. 2). This allowed us to combine the strain RZ2MS9 with Ab-
V5 as well as RZ2MS16 with Ab-V5 for the field trial.
3.2. Effects of PGPB inoculation on maize growth
We assessed the effects of bacterial inoculation on several parameters
of maize growth under field conditions. The treatments RZ2MS9 + Ab-
V5 and Ab-V5 significantly increased plant height by 2.8% and 2.6% and
stalk diameter by 9% and 6.9%, respectively, when compared to the
non-inoculated control (Fig. 1A and C). We also observed that the
treatments RZ2MS9 + Ab-V5 and RZ2MS9 had no significant effect on
ear height as compared to the control. However, these treatments
increased ear height when compared to the treatment RZ2MS16
(Fig. 1B). RZ2MS16 + Ab-V5 significantly reduced the stalk diameter

Fig. 1. Effects of bacterial inoculation on maize growth at 60 days after seeding: Plant height (A); Ear height (B); Stalk diameter (C); Number of leaves (D); Cob size
(E); Weight of 100 grains (F); Number of cobs (G); Grain yield (H). Different letters indicate statistically significant differences among means (LSD test, p-value <
0.05). RZ2MS16 = Burkholderia ambifaria strain RZ2MS16; RZ2MS16 + Ab-v5 = Burkholderia ambifaria strain RZ2MS16 and Azospirillum brasilense strain Ab-v5; Ab-
v5 = Azospirillum brasilense strain Ab-v5; RZ2MS9 = Bacillus thuringiensis strain RZ2MS9; RZ2MS9 + Ab-v5 = Bacillus thuringiensis strain RZ2MS9 and Azospirillum
brasilense strain Ab-v5; Control = non-inoculated control.

4
J.A. Ferrarezi et al.
when compared to RZ2MS9 + Ab-V5, Ab-V5, and RZ2MS16 (Fig. 1C). In
addition, Ab-V5 significantly increased the number of leaves by 9.7%,
7.8%, 12.8%, and 14.1% when compared to the control, to RZ2MS16, to
RZ2MS9 + Ab-V5, and to RZ2MS16 + Ab-V5, respectively (Fig. 1D).
Finally, the treatment RZ2MS16 + Ab-V5 significantly reduced plant
height and ear height in comparison with the control (Fig. 1A and B).
For the maize productivity parameters, we observed that the treat­
ments Ab-V5, RZ2MS9 + Ab-V5, RZ2MS16, and RZ2MS9 did not have
any effect on cob size when comparing with the control, but these
treatments significantly increased cob size when comparing with
RZ2MS16 + Ab-V5 (Fig. 1E). The treatments RZ2MS16 + Ab-V5, Ab-V5,
and RZ2MS16 significantly increased the weight of 100 grains by
13.75%, 13.66%, and 10.43%, respectively, against the control
(Fig. 1F). Additionally, the treatment RZ2MS16 + Ab-V5 significantly
reduced the number of cobs (− 19.7%) in contrast to the control
(Fig. 1G). Nevertheless, the treatments did not statistically differ from
each other for the estimated average yield (Fig. 1H).
3.3. Bacterial community abundance
The treatments had no significant effect on the bacterial abundance
associated with maize leaves and roots. The bacterial abundance
detected in the leaf samples were similar among the treatments, but
lower than in the roots, except for the treatments RZ2MS16 and
RZ2MS16 + AbV5 (Supplementary Fig. 3). Considering only the plant
niches, roots presented significantly higher bacterial abundance as
compared to the leaves (Supplementary Table 1).
3.4. Bacterial community diversity and structure
The alpha-diversity estimated by Chao1 index significantly differed
according to the plant niches. Rhizosphere samples presented higher
bacterial richness than root and leaf samples (Fig. 2A). On the other
hand, similar values of Chao1 richness index were observed among
treatments (Fig. 2B), indicating that the maize-associated bacterial
community responded to niche variation but not to the inoculation
treatments. More information about the bacterial richness and diversity
Fig. 2. Alpha-diversity estimated by using Chao1 index for plant niches (A), and for the treatments (B). RZ2MS16 = Burkholderia ambifaria strain RZ2MS16;
RZ2MS16 + Ab-v5 = Burkholderia ambifaria strain RZ2MS16 and Azospirillum brasilense strain Ab-v5; Ab-v5 = Azospirillum brasilense strain Ab-v5; RZ2MS9 = Bacillus
thuringiensis strain RZ2MS9; RZ2MS9 + Ab-v5 = Bacillus thuringiensis strain RZ2MS9 and Azospirillum brasilense strain Ab-v5; Control = non-inoculated control.
5
Applied Soil Ecology 170 (2022) 104297
indices assessed can be found in the Supplementary Table 1.
PCoA displayed the bacterial community structure associated to
maize samples. No significant dissimilarities were observed among the
treatments, but clear differences in the bacterial community structure
were detected when comparing the maize niches (Fig. 3). However, we
could observe a slight separation of the samples belonging to the
treatment RZ2MS16 + AbV5 in the root (Fig. 3).
Finally, according to the PCA, a direct and positive correlation was
observed between the variables stalk diameter and plant height, which
clustered together close to PC1, corresponding to 43.9% of the samples'
variability. A null correlation was detected between the variables ear
height and number of leaves (Supplementary Fig. 4).
3.5. Bacterial community taxonomic composition
The sequencing of bacterial communities associated with maize leaf,
root, and rhizosphere soil samples generated a total of 2,409,067 high
quality sequences with an average of 133,837 reads per treatment per
niche (Supplementary Table 2). After data processing, 5861 ZOTUs were
obtained and these sequences were classified into 25 different classes.
The predominant classes were Betaproteobacteria, Gammaproteobac­
teria, Actinobacteria, Alphaproteobacteria, and Cytophagia (Fig. 4).
The treatment RZ2MS16 seems to have increased the RA of Gam­
maproteobacteria and decreased the RA of Actinobacteria in leaf tissues
when compared to the control. Other treatments had no effect over the
RA of bacterial taxa in the community (Fig. 4). We observed that the
bacterial communities associated with maize roots presented increased
relative abundance (RA) of Gammaproteobacteria and lower RA of
Betaproteobacteria when treated with RZ2MS16 and RZ2MS16 + AbV5,
as compared to the non-inoculated control (Fig. 4). Rhizosphere samples
presented homogeneous RA of bacterial taxa among treatments, indi­
cating that the bioinoculant treatments did not affect the bacterial
community associated with this plant niche (Fig. 4).
In the leaf core microbiome, four bacterial classes were identified:
Gammaproteobacteria, Actinobacteria, Alphaproteobacteria, and Cyto­
phagia (Fig. 5A). In root, seven classes were identified: Gammaproteo­
bacteria, Actinobacteria, Alphaproteobacteria, Bacilli, Cytophagia, and
J.A. Ferrarezi et al. Applied Soil Ecology 170 (2022) 104297

Fig. 3. Principal coordinate analysis (PCoA) dis­

playing the structure of the bacterial community
associated with maize leaf (in red), root (in green),
and rhizosphere (in blue). RZ2MS16 = Burkholderia
ambifaria strain RZ2MS16; RZ2MS16 + Ab-v5 =
Burkholderia ambifaria strain RZ2MS16 and Azospir­
illum brasilense strain Ab-v5; Ab-v5 = Azospirillum
brasilense strain Ab-v5; RZ2MS9 = Bacillus thur­
ingiensis strain RZ2MS9; RZ2MS9 + Ab-v5 = Bacillus
thuringiensis strain RZ2MS9 and Azospirillum brasi­
lense strain Ab-v5; Control = non-inoculated control.

Fig. 4. Taxonomic profile, at the class level, of the bacterial community of maize according to the different plant niches (Leaf, Root, and Rhizosphere). The group
“Others” plots the bacterial groups with counts < 10 based on total counts. RZ2MS16 = Burkholderia ambifaria strain RZ2MS16; RZ2MS16 + Ab-v5 = Burkholderia
ambifaria strain RZ2MS16 and Azospirillum brasilense strain Ab-v5; Ab-v5 = Azospirillum brasilense strain Ab-v5; RZ2MS9 = Bacillus thuringiensis strain RZ2MS9;
RZ2MS9 + Ab-v5 = Bacillus thuringiensis strain RZ2MS9 and Azospirillum brasilense strain Ab-v5; Control = non-inoculated control.

6
J.A. Ferrarezi et al. Applied Soil Ecology 170 (2022) 104297

Fig. 5. Heat map of the relative abundance and prevalence of maize core microbiome, at the class level, according to the different plant niches: leaf (A), root (B), and
rhizosphere (C). Only taxa contributing to ≥0.1% of relative abundance were selected for the heat map, which were represented in decreased order of prevalence.
Color legend and scale are provided in the figure.

Sphingobacteria (Fig. 5B). In rhizosphere, the most abundant bacterial
classes were Betaproteobacteria, Alphaproteobacteria, Actinobacteria,
Bacilli, Nitrospira, Deltaproteobacteria, Gammaproteobacteria, and
three groups (Gp1, Gp3 e Gp5) of Acidobacteria (Fig. 5C).
3.6. Analysis of differentially abundant bacterial taxa
The linear discriminant analysis (LDA) effect size (LEfSe) was used to
identify the bacterial taxa that significantly differed in abundance
among treatments inside each plant niche. In leaf samples, 4 bacterial
groups were differentially abundant among the treatments (p-value <
0.05, LDA score > 2.0) (Fig. 6A). Three bacterial groups were signifi­
cantly enriched in leaf samples of plants treated with RZ2MS16 + Ab-
V5, including the class Actinobacteria, the phylum Actinobacteria, and
the order Actinomycetales (Fig. 6A). One bacterial group (Lactoba­
cillales order) was significantly enriched in leaf samples of plants treated
with RZ2MS9 + Ab-V5. For root samples, the class Actinobacteria, the
phylum Actinobacteria, and the order Actinomycetales were also
enriched in plants treated with RZ2MS16 + Ab-V5, while the families
Bradyrhizobiacea and Alcaligenaceae were enriched in the roots of

7
J.A. Ferrarezi et al. Applied Soil Ecology 170 (2022) 104297

Fig. 6. Linear discriminant analysis (LDA) effect size (LEfSe) computed for bacterial differentially abundant ZOTUs among the treatments grouped by niche (leaf,
root, and rhizosphere). Significant differences are defined at p-value < 0.05 and a LDA score > 2.0. RZ2MS16 = Burkholderia ambifaria strain RZ2MS16; RZ2MS16 +
Ab-v5 = Burkholderia ambifaria strain RZ2MS16 and Azospirillum brasilense strain Ab-v5; Ab-v5 = Azospirillum brasilense strain Ab-v5; RZ2MS9 = Bacillus thuringiensis
strain RZ2MS9; RZ2MS9 + Ab-v5 = Bacillus thuringiensis strain RZ2MS9 and Azospirillum brasilense strain Ab-v5; Control = non-inoculated control.

plants treated with RZ2MS16 (Fig. 6B). Finally, we detected that the
bacterial class Actinobacteria, the order Actinomycetales, and the family
Intrasporangiaceae were significantly enriched in rhizosphere samples
from maize plants treated with RZ2MS9 + Ab-V5 (Fig. 6C). Also, the
class Alphaproteobacteria was significantly enriched in rhizosphere
samples collected from plants treated with RZ2MS9 (Fig. 6C).
3.7. Functional analyses
We observed significant differences in the functional categories
“Respiration” and “Solubilization of Cu, Mg, and K” among the bacterial
treatments used in this study (Fig. 7). Rhizosphere soil samples from
plants treated with RZ2MS16 presented the highest respiration capacity,

8
J.A. Ferrarezi et al.
Fig. 7. Differential abundance of predicted genes related to respiration (A), and solubilization of Cu, Mg, and K (B) among treatments. RZ2MS16 = Burkholderia
ambifaria strain RZ2MS16; RZ2MS16 + Ab-v5 = Burkholderia ambifaria strain RZ2MS16 and Azospirillum brasilense strain Ab-v5; Ab-v5 = Azospirillum brasilense strain
Ab-v5; RZ2MS9 = Bacillus thuringiensis strain RZ2MS9; RZ2MS9 + Ab-v5 = Bacillus thuringiensis strain RZ2MS9 and Azospirillum brasilense strain Ab-v5; Control =
non-inoculated control.
while rhizosphere samples treated with RZ2MS9 presented the lowest
capacity for the same attribute (Fig. 7A). Soil from plants treated with
RZ2MS16 also showed the highest capacity for the functional category
“Solubilization of Cu, Mg, and K”, while Ab-V5, RZ2MS9, and RZ2MS9
+ Ab-V5 presented similarly low capacity for this attribute (Fig. 7B). The
functional categories were then associated with the production param­
eters using NMDS (Fig. 8). We noticed the following close associations:
(1) among the solubilization of heavy metals, cob size, and stalk diam­
eter; (2) between salicylic acid production and plant height, (3) among
organic sulfur metabolism, iron reduction, number of cobs, number of
leaves, and cob height; (4) between calcium precipitation and the
weight of 100 grains (Fig. 8).
4. Discussion
The successful application of bacterial inoculants to enhance plant
productivity relies mainly on the ability of microorganisms that show
promising results in laboratory and greenhouse trials to overcome bar­
riers and retain their characteristics when applied in the field (Sessitsch
et al., 2019). Analysing the effect of biological products on the plant-
associated microbiota as well as their compatibility with products
adopted by the farmers is essential to understand the persistence and
efficacy of the introduced microbes.
Many PGPB have been reported as successfully promoting the
growth of plants in controlled conditions (Hu et al., 2017; Maqsood
et al., 2021), including with maize as the host plant (Aydinoglu et al.,
2020; Batista et al., 2018; Ullah et al., 2020). Fewer studies have
extended PGPB assays to field conditions (Fukami et al., 2016; Hungria
et al., 2010, 2015, 2016; Marks et al., 2015). In this study we analysed
the effects of the PGPB Burkholderia ambifaria RZ2MS16, Bacillus thur­
ingiensis RZ2MS9, and Azospirillum brasilense Ab-V5, as well as of two
bacterial combinations (RZ2MS16 + Ab-V5 and RZ2MS9 + Ab-V5) on
the growth of maize (hybrid DKB 390) in a field trial.
To compose the consortia treatments used in this study, we first
examined the compatibility among the PGPB strains to be tested and no
negative interactions were detected. Ab-V5 has been used in co-
inoculation with other bacteria, including Azospirillum brasilense Ab-
V6 and Bradyrhizobium spp. (Hungria et al., 2010, 2015, 2016). To the
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Applied Soil Ecology 170 (2022) 104297
best of our knowledge this is the first study to evaluate Ab-V5 strain
performance alone or in combination with Burkholderia ambifaria and
Bacillus thuringiensis.
Here, we observed that the RZ2MS9 + AbV5 consortia significantly
increased maize plant height and stalk diameter in comparison with
non-inoculated control and the other treatments. Moreover, PCA ana­
lyses indicated that stalk diameter and plant height had a strong and
positive correlation between each other, suggesting that plant height
increase is in great part related to stalk diameter increase. In fact, the
superiority of consortia over single strains is well known since several
studies suggest that more complex consortia can provide plants with
increased disease resistance and growth promotion effects as compared
to single bacterial strains (Armanhi et al., 2018; Hungria et al., 2010,
2015, 2016; Lopes et al., 2021). In addition, recent studies have reported
bacterial strains that show little or no effects as single inoculants but
exhibit plant growth promotion effects when used in a consortium
(Armada et al., 2016; Bai et al., 2003; Khan et al., 2020; Mishra et al.,
2009; Zhikang et al., 2021).
Here, our treatments have slightly affected maize growth, with
inferior increases to those reported by Batista et al. (2018) with inocu­
lation of RZ2MS9 and RZ2MS16 in maize in controlled conditions. The
authors reported an increase of up to 40% on the maize shoot dry weight
when inoculated with these two PGPB (Batista et al., 2018). The effect of
the inoculation with the strain Ab-V5 in our study was also inferior to
that reported by Hungria et al. (2010) in field conditions, where in­
creases of up to 25% in the wheat and maize biomass were observed
when treated with Ab-V5 and Ab-V6. It is important to clarify that
although the study conducted by Hungria et al. (2010) was carried out in
field conditions, the Ab-V5 strain was tested only in co-inoculation with
other Azospirillum strain Ab-V6. Furthermore, plant genotype (Compant
et al., 2020; Cregger et al., 2018), soil properties (Florio et al., 2017),
diverse environmental or climatic conditions, agricultural management
practices, and land use (Saad et al., 2020) can also be important sources
of variation. Thus, different outcomes can be expected.
Although reports on enhancement of plants growth through PGPB
inoculation are widely available, there has been high variability and
inconsistency in the results of studies carried out in laboratory, green­
house, and field conditions (Gouda et al., 2018; Saad et al., 2020). For
J.A. Ferrarezi et al. Applied Soil Ecology 170 (2022) 104297

Fig. 8. Non-metric multidimensional scaling (NMDS) ordination of the metabolic pathways identified through functional prediction. Distribution of samples by
treatment group and production parameters vectors determined by envfit (A). Distribution of metabolic pathways against the production parameters vectors (B).

instance, field experiments using Azospirillum spp. and other PGPB have
shown increases in maize grain yield ranging from 5 to 75% (Marks
et al., 2015). The variable performance of bacterial inoculants under
field conditions may be due to uncontrolled abiotic and biotic factors
that can affect the growth and performance of the applied microbes
(Saad et al., 2020). Some studies have also reported the influence of
plant genotype on the degree of beneficial response to PGPB inoculation,
including with A. brasilense Ab-V5 (Vidotti et al., 2019).
We also investigated the impact of the microbial treatments on the
abundance, diversity, and structure of the leaf-, root-, and rhizosphere-
associated bacterial communities. We observed that the inoculation did
not affect the bacterial abundance in the maize rhizosphere, leaves, or

10
J.A. Ferrarezi et al.
roots. Similar results were found by Estrada-Bonilla et al. (2021) when
applying compost and phosphate-solubilizing bacteria to sugarcane in a
greenhouse experiment. The authors found only a small effect in the
diversity of microbes in soil samples, although inoculation shifted the
soil bacterial community structure. Also, like other studies, we observed
that microbial richness and structure differed among plant-associated
niches (Cregger et al., 2018; Dickey et al., 2020). Previous studies
have proposed that the occurrence, abundance, and activities of indi­
vidual bacterial taxa depend on the microenvironment provided by the
plant compartment, meaning that the niche adaptation may have a
major role in selectively filtering and recruiting different microorgan­
isms (Compant et al., 2020; Trivedi et al., 2020).
Our results confirmed previous findings that the roots and leaves
harbor a lower diversity and distinct microbial communities as
compared to bulk soil or rhizosphere (Kudjordjie et al., 2019). In the
present study, the bacterial richness in the maize rhizosphere was
greater than that in the roots, which was greater than that in the leaves.
The same pattern was observed in Populus trees by Cregger et al. (2018),
who also reported that both archaeal/bacterial and fungal community
composition shifted more across habitats than among tree genotypes.
Peiffer et al. (2013) analysed 26 different maize genotypes and observed
that a large proportion of microbial diversity among maize inbred ge­
notypes was field-specific. Furthermore, Dickey et al. (2020) found
variation in alpha diversity of microbial communities in rhizosphere
samples when collected at different depths, where the richness
decreased with increasing sampling depth. Thus, our findings corrobo­
rate the knowledge of the niche-mediated plant microbiota structure
and possible low heritability between field trials.
In the present study, the more abundant bacterial classes in the core
microbiome were Betaproteobacteria, Alphaproteobacteria, Gammap­
roteobacteria, and Actinobacteria. This finding is in accordance with
other studies (Cregger et al., 2018; Gouda et al., 2018; Pascale et al.,
2020; Trivedi et al., 2020). Although microbial composition varies
throughout plant development, a small fraction of microbial taxa
belonging to the core microbiota are consistently maintained in high
relative abundances throughout plant development (Trivedi et al.,
2020). Noteworthy, in our study, the community composition of these
classes remained similar in different plant-associated niches, indicating
a conservative adaptation to the plant environment.
Taxonomy analysis revealed which ZOTUs (at genus level) are more
strongly associated with each host-niche within the maize plant. How­
ever, due to the high microbial diversity found in the plant tissues and
the high throughput sequencing resolution of this study, more mean­
ingful interpretations are given at higher taxonomic levels. It has been
reported that seed-borne microorganisms preferentially become associ­
ated with aboveground plant tissues, whereas soil-derived microorgan­
isms are mainly associated with the rhizosphere and roots (Torres-Cortés
et al., 2018).
The highly abundant Proteobacteria group, especially Betaproteo­
bacteria, Alphaproteobacteria, and Gammaproteobacteria, detected in
maize roots and rhizosphere samples in this study, has been reported to
be commonly associated with soil and/or plant roots of several plant
species (Chauhan et al., 2011; Johnston-Monje et al., 2016; Kudjordjie
et al., 2019) including maize (Niu et al., 2017; Peiffer et al., 2013).
Proteobacteria are well known to respond to labile carbon sources and
are generally considered to be fast-growing microbes, whose pop­
ulations fluctuate opportunistically (Peiffer et al., 2013). Corroborating
our results, Cotton et al. (2019) and Niu et al. (2017) also found Acti­
nobacteria as one of the main bacterial classes prevailing in maize
rhizobiome.
In comparison with belowground tissues, the richness in the cereal
phyllosphere was low especially considering the low relative abundance
of Betaproteobacteria. This could be related to limited nutrient re­
sources in leaves and harsh environmental conditions, such as high ra­
diation and temperature fluctuations (Barrera et al., 2019). We found
that the classes Gammaproteobacteria, Actinobacteria, and
11
Applied Soil Ecology 170 (2022) 104297
Alphaproteobacteria were the most abundant bacterial groups associ­
ated with maize leaves, which is consistent with the phyllosphere mi­
crobial composition of many plant species (Bodenhausen et al., 2014;
Müller et al., 2016; Trivedi et al., 2020; Vorholt, 2012). The PGPB
RZ2MS16 slightly affected the class Gammaproteobacteria but not the
class Actinobacteria in leaf and the class Betaproteobacteria in root,
indicating a factor that should be better investigated.
The linear discriminant analysis (LDA) identified a differential
abundance of the class Actinobacteria and the order Actinomycetales
(class Actinobacteria) in leaf and root of plants treated with RZ2MS16 +
Ab-V5. The same bacterial group was enriched in rhizosphere with the
treatment RZ2MS9 + Ab-V5, including the family Intrasporangiaceae
(class Actinobacteria). Also, the class Alphaproteobacteria was enriched
in rhizosphere samples collected from plants treated with RZ2MS9. In
maize plants, Alphaproteobacteria, Actinobacteria, and Gammaproteo­
bacteria are generally the most abundant classes (Barrera et al., 2019;
Müller et al., 2016). In addition, certain Gammaproteobacteria have
been shown to be plant protective agents (Bodenhausen et al., 2014) and
their presence in maize leaves could be due to its mobility towards
nutrient sites on the leaf, with significant advantages over immobile
bacteria (Barrera et al., 2019).
The order Lactobacillales (class Bacilli) was enriched in leaf samples
from plants treated with RZ2MS9 + Ab-V5. Also, the families Bra­
dyrhizobiacea (class Alphaproteobacteria) and Alcaligenaceae (order
Burkholderiales) were enriched in the roots of plants treated with
RZ2MS16. The differential occurrence of the order Lactobacillales (class
Bacilli) in leaf samples and Burkholderiales in root samples was previ­
ously described by Given et al. (2020) in wild and bait plants of Oxyria
digyna (Mountain sorrel). The authors reported that the Bacilli class was
highly enriched in the leaf samples. In contrast, several OTUs repre­
senting Burkholderiales were more abundant in the root communities.
Also, based on our results, the inoculation did not modify the bac­
terial community associated with maize. One explanation may be the
excessive overlap between the resident and introduced communities.
Possibly, the introduced microbes were not able to outcompete existing
microbial taxa and to use available resources to grow and spread. In fact,
the higher the number of vacant niches, the higher the chances of the
inoculant to successfully establish in their new habitat. Also, the high
level of overlap between the resident community and the niche of an
inoculated bacteria reduces invasion success (Mawarda et al., 2020;
Yang et al., 2017).
However, it is hard to ensure that the sample collection for micro­
biome analyses in this study was done at the right time to detect any
inoculant impact on the maize microbiome. A few studies have accessed
inoculation effects over time. Some of them have reported impacts on
microbiome several months after inoculation (Wang et al., 2018; Yin
et al., 2013), others only few days after inoculation (Johansen and
Olsson, 2005). For instance, Johansen and Olsson (2005) found that
Pseudomonas fluorescens DR54 affected the structure of rhizosphere
microbiome associated with barley up to 6 days after inoculation but,
after 9 days, it returned to its original structure. Recently, Mawarda
et al. (2020) published a meta-analysis study to assess whether microbial
inoculants alter soil microbial community composition. The authors
found that, from 26 studies using high-throughput sequencing, over 96%
reported that microbial application led to changes in the native micro­
bial community composition. However, from the 78 reviewed studies
that used profiling methods, 82% detected an impact following inocu­
lation whereas 18% did not report any significant effect.
The variability among studies over the inoculant impact on plant and
soil microbiome might also be correlated with the inoculant concen­
tration, the method of application, and the diverse environmental con­
ditions, including the soil type (Florio et al., 2017; Fukami et al., 2016;
Marks et al., 2015). Florio et al. (2017) reported contrasting effects of
inoculation with denitrifiers PGPB between different soil types. Fukami
et al. (2016) tested the inoculant application in-furrow, via soil spray at
sowing, and via leaf spray after seedlings emergence, in comparison to
J.A. Ferrarezi et al.
seed inoculation. Different leaf N contents were detected among treat­
ments, and inhibitory effects of the soil spray inoculation treatment were
observed. Marks et al. (2015) compared the inoculation via leaf spray
and seed inoculation with and without lipo-chitooligosaccharides
(LCOs) metabolites. The authors found that, when the inoculant was
applied alone on the seeds, significant increases in maize grain yield
were observed in two out of six experiments. However, when supple­
mented with LCO-enriched metabolites, the effect of the inoculation
method seemed to be reduced and significant increases in grain yield in
comparison to the non-inoculated control were observed in five out of
six field experiments for both leaf spray and seed inoculation strategy.
It is important to add to this complex scenario that the plant
microbiome composition varies along the plant life cycle (Edwards et al.,
2018). The microbial communities can be highly dynamic in the early
vegetative phase, then begins to converge throughout vegetative growth
and stabilizes during the reproductive phase. Further studies are needed
and should consider evaluating the impact of inoculation over time and
using more than one plant genotype, as the plant genotype is also an
important source of variation for the plant-associated microbial com­
munities (Compant et al., 2020; Cregger et al., 2018).
In our trial, the potential microbial soil respiration and solubilization
of some macro and micronutrients were estimated by using the abun­
dance of functional predicted genes related to these parameters. Soil
respiration is an important indicator of soil fertility and biological ac­
tivity, which in turn impacts plant yield (Bojarszczuk et al., 2019). A
study with maize correlated soil respiration with the plant's biological
activity and biochemical parameters. The results showed that soil
respiration had a positive correlation with the grain and straw yield of
maize and a negative correlation with evapotranspiration and soil
moisture (Bojarszczuk et al., 2017).
The abundance of functional predicted genes related to solubiliza­
tion of Cu, Mg, and K shows the potential of the plant to keep biological
and biochemical reactions functioning properly (Ribeiro et al., 2020).
Copper is a cofactor for several metalloproteins and other macromole­
cules involved in essential metabolic processes, including cell wall
lignification, respiration, photosynthesis, and various protection mech­
anisms (Abdel Latef et al., 2020; Nazir et al., 2019). Magnesium is an
essential element for the formation of the chlorophyll molecule, a
cofactor for many enzymatic processes, and acts as a binding element for
the combination of ribosome subunits that are mandatory for the for­
mation of proteins (Gaj et al., 2018; Guo et al., 2016; Zhang et al., 2020).
Also, when potassium is lacking, plants present poorly developed roots
and small seed production and are more susceptible to diseases and
insects attacks (Meena et al., 2018).
The association with the plant production parameters and functional
gene categories showed a positive association between salicylic acid
production, plant height, and dry matter. Previous work, carried out by
Hussein et al. (2007) shows that maize plants irrigated with salicylic
acid increased the production of all amino acids (except methionine),
promoting the development of all plant-growth parameters (plant
height, number and area of green leaves, stem diameter, and the dry
weights of the stem, leaves, and whole-maize plant), corroborating our
results. Another relevant and positive association found is the one be­
tween calcium precipitation and the weight of 100 grains. Calcium
concentration in maize tissues at physiological maturity was reported by
Szczepaniak et al. (2016) as a diagnostic tool to evaluate yield perfor­
mance during the growing season. These authors found that the higher
the Ca concentration in the grain, the lower the maize yield, drawing a
parallel with the number of kernels in the cob.
An association among organic sulfur metabolism, iron reduction,
number of cobs, number of leaves, and ear height was also noticed.
Previous studies show sulfur as a key element for the formation of
proteins, along with nitrogen, contributing significantly to the increase
of maize yield (Carciochi et al., 2020; Liu et al., 2021; Sutar, 2017).
Sulfur also plays a key role in chlorophyll formation and oil synthesis
(Sutar, 2017). Also, it is not clear if the positive association among the
12
Applied Soil Ecology 170 (2022) 104297
solubilization of heavy metals, cob size, and stalk diameter is the result
of a biochemical connection or if it is just a random occurrence.
5. Conclusions
Our study contributes to understanding the efficacy of microbial
inoculants for maize in field conditions and their effect on the maize
native microbiome. The inoculation did not significantly alter the plant-
associated microbiome and had no remarkable effect on maize growth.
Therefore, our findings add evidence to the lack of consistent efficacy of
microbial inoculants when scaled up from controlled conditions,
whether in the laboratory or greenhouse, to a complex field trial.
Moreover, assessments over time may be better suited to investigate the
inoculants fate and their impact on the microbiome associated with
maize when grown under field conditions. We believe that this knowl­
edge can be applied to improve the development process of microbial
products for agricultural use, considering the need for inoculants whose
effects are persistent and replicable under field conditions.
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.apsoil.2021.104297.
CRediT authorship contribution statement
PACE: Writing – Original Draft, Writing- Reviewing, and Editing.
JAF: Formal analysis, Writing – Reviewing, and Editing. BDB: Investi­
gation, Writing – Reviewing, and Editing. RMA: Investigation, Writing –
Reviewing, and Editing. BAPT: Investigation, Writing – Reviewing, and
Editing. PAMA: Formal analysis, Writing – Reviewing, and Editing. BML:
Formal analysis, Writing – Reviewing. MLB: Investigation, Formal
analysis, Writing – Reviewing. EJO: Formal analysis, Writing –
Reviewing, and Editing. JLA: Writing – Reviewing, and Editing. MCQ:
Conceptualization, Supervision, Writing – Reviewing, and Editing.
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.
Acknowledgements
We thank the Department of Genetics in the name of Dr. Roberto
Fritsche-Neto for providing the maize seeds used in this study. We also
thank Dr. Mariangela Hungria, researcher at Embrapa Soja, for
providing the commercial bioinoculant Ab-V5 and Dr. Itamar Soares de
Melo and Dr. Rodrigo Gouveia Taketani from Embrapa Meio Ambiente
for sequencing support.
Funding
We thank São Paulo Research Foundation – FAPESP for the grants
(Proc. No. 2015/01188-9 and 2019/04697-2) and for the fellowship to
JAF (Proc. No. 2019/25720-2). We thank the National Council for Sci­
entific and Technological Development - CNPq for the fellowships
granted to PACE, RMA, PAMA, BDB and BML and to also Coordination
for the Improvement of Higher Education Personnel-CAPES for the
fellowship granted to BAPT and MLB.
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