Rhizobia and Azospirilla Co-Inoculation
Boosts Growth and Productivity of Common Bean
Matheus Messias1*, Princewill Chukwuma Asobia1,2
and Enderson Petrônio de Brito Ferreira3
1Postgraduate
Program in Agronomy, Federal University of Goiás, Goiânia, Goiás, Brazil
2Michael Okpara
University of Agriculture, Abia, Umudike, Nigeria
3Embrapa Arroz e
Feijão, Santo Antônio de Goiá, Goiás, Brazil
*For correspondence: messyas023@gmail.com
Received 29 July 2022; Accepted
02 December 2022; Published 30 December 2022
Abstract
The rhizobia and azospirilla
combined use is an alternative to N-fertilizers, besides to guarantee high
grain yields for the crops. In this work we evaluated the effect of on seed
rhizobia and on aerial part azospirilla co-inoculation on some selected
production parameters of common bean. A random block design with four
replicates was used for the field experiments. The evaluated treatments were:
1- AC- Absolute control; 2- NfT- Nitrogen fertilization; 3- Rt; 4- Rt + Ab1l;
5- Rt + Ab2l; 6- Rt + Ab3l; 7- Rt + Ab4l and 8- RP- Registered product. Rhizobium tropici (Rt) was applied using
2 doses ha-1 and, Azospirillum
brasilense (Ab) was applied using 1, 2, 3 or 4 doses ha-1 at V2/V3
phenological stage, while the RP treatment received two 2 ha-1 of R.
tropici and 3 doses ha-1 of A. brasilense at V2/V3
phenological stage. Nodulation (nodule number – NN, nodule dry weight –
NDW); growth (shoot dry weight – SDW, root dry weight - RDW) and, productive
(number of pods – NP; number of grains - NG, and grain yield - GY) parameters
were evaluated. The Rt+Ab3l co-inoculation provided an increase in NN, MNS, NV
and NG, in addition to presenting the highest GY stability, producing about
3439 kg ha-1 as an average from the five locations, which represents
an increase of 100, 93, 74 and 83 kg ha-1 in comparison to TN, PR,
Rt, and TA, respectively. © 2022 Friends Science Publishers
Key words: Biological nitrogen fixation; Plant-bacteria
interaction; Symbiosis; Inoculation; Co-inoculation
Nitrogen fertilizers are crucial for the development and attainment of
high yields in the common bean crop (Lacerda et al. 2019). However, the
excessive use of nitrogen fertilizers is quite common among producers, who
apply doses higher than 120 kg ha-1 of N (Peres et al. 2018).
Under these conditions, nitrogen fertilizers pose risks to the environment,
mainly through the emission of greenhouse gases, in addition to the high costs
and losses through leaching and volatilization (Souza and Ferreira 2017).
Co-inoculation is a viable alternative to
replacing nitrogen fertilization, characterized by the associated use of plant
growth-promoting microorganisms, exhibiting different mechanisms of action,
such as the association of the bacteria Rhizobium
tropici and Azospirillum brasilense.
Co-inoculation results in a synergistic effect, which provides a greater
stimulus compared to the effect of each mechanism of action alone (Hungria et
al. 2013). Azospirillum bacteria are strongly related to the
production of phytohormones, such as indole acetic acid (Masciarelli et al.
2013), gibberellins (Lenin and Jayanthi 2012), cytokinins and ethylene
(Strzelczyk et al. 1994), providing better plant development, greater
absorption of nutrients, increased tolerance to water deficit
(Huergo et al. 2008), due to the increase in the exploited soil area
triggered by the greater root development (Puente et al. 2018).
The co-inoculation is an environmentally
friendly option, which can partially or totally replace the use of
N-fertilizers inputs. This fact was highlighted in the study conducted by
Bettiol et al. (2021), where co-inoculation increased the grain yield by 187.75 kg ha-1
in camparison with the nitrogen fertilization, thus demonstrating the ability
of co-inoculation to replace nitrogen fertilizers in common bean cultivation.
In general, seed inoculation is
the most used form of application of plant growth-promoting rhizobacteria
(PGPR). However, considering the
possibilities of inoculation via seed, furrow, or foliar, there are different technical
recommendations for carrying out co-inoculation. In those where R. tropici is applied via seed and A. brasilense via spraying the plants, the best results were
observed with the highest concentration of A.
brasilense (Souza and Ferreira 2017). These authors stated that the
application of three doses of A.
brasilense via foliar resulted in production increases equivalent to 278
and 560 kg ha-1 in comparison to treatments with nitrogen
fertilization and inoculation only with R.
tropici, respectively. Azospirillum
foliar spraying is an alternative that allows microorganisms to interact with
the phyllosphere, altering the dynamics and composition of plant hormones
(Gonzalez-Lopez et al. 1991; Filipini et al. 2021). The use of
PGPR's foliar spraying increased the common bean production (Souza and Ferreira
2017), corn biomass production (Portugal et al. 2016; Filipini et al.
2021) and improved soybean nodulation (Puente et al. 2018; Filipini et
al. 2021).
Knowing the benefits of A.
brasilense as a plant growth promoter and its help in N fixation
improvement when associated with R. tropici, it is essential to study
different forms and doses of Azospirilla application in field experiments. Thus, this
work aimed to determine the effect of the co-inoculation of rhizobia and
different doses of azospiilla applied via
foliar spraying over nodulation, growth and grain production parameters of the
common bean cropped under field conditions in commercial farms.
Five field experiments were
performed in three different cropping seasons (winter 2018, water 2018–2019,
winter 2019, and water 2019–2020) in different areas, to evaluate the
efficiency of the treatments in different soil and climatic conditions,
cultural treatments, and technological levels of each experimental area.
Detailed information is presented in Table 1.
For the chemical analysis of the
soil, 10 soil subsamples were taken from each experimental area at 0–20 cm
depth before sowing. The subsamples were homogenized to generate a composite
sample used to determine the chemical characteristics: Soil pH; Exchangeable
Ca, Mg, and Al; P, K, Cu, Zn, Fe and Mn contents and, soil organic matter according
to Donagema et al. (2011). The soil analysis results are presented in
Table 2.
Commercial inoculants formulated
separately with SEMIA 4077 strain of R.
tropici and Ab-V5 strain of A.
brasilense were used: 1) Biomax Premium turfoso Feijão, containing R. tropici in a
concentration of 2x109 CFU mL-1 (Colony Forming unit) and
2) Biomax Premium Azum, containing A.
brasilense in a concentration of 3x108 CFU mL-1. Both
inoculants were provided by Vittia Fertilizantes e Biológicos Ltda. These
inoculants were compared with the inoculant registered for the common bean
crop, provided by Stoller do Brasil Ltda, as follow: Masterfix Feijão
(turfoso), containing R.
tropici in a concentration of 2x109 CFU g-1 and
Masterfix Gramíneas (liquido), containing A.
brasilense in a concentration of 2x108
CFU mL-1.
Eight treatments were evaluated,
consisting of a inoculation only with R.
tropici (Rt), four combinations of R.
tropici and A. brasilense doses,
an absolute control (AC - without co-inoculation and without N fertilization),
a control N-fertilization (NfT - without co-inoculation), and the registered
product (RP). In N-fertilizer treatment (NfT), 80 kg ha-1 of N were
used, with 20 kg ha-1 of N applied at sowing and 60 kg ha-1
of N applied at the V4 stage using urea. Complementary information
about the eight treatments is given in Table 3.
Experiments were performed under
field condictions on a random block design with 4 repetitions. The experiments were installed in plots composed of six four-meter-long rows,
spaced by 0.45 m between rows, totaling 10.8 m² per plot.
Aiming to lead the base soil
saturation to 70% and the soil pH to 5.5, limestone was applied 50 days before
the experiment settlement, according to the needs of each location as shown in
Table.
The fertilization with
phosphorus (P2O5) and potassium (K2O) was done
in the sowing operation, according to the needs indicated by soil analysis.
According to the results of the soil chemical analysis and the technical
recommendation for the common bean crop (Carvalho and Silveira 2021), the
experiments conducted in Formosa-GO/2018–19 and Cristalina-GO/2019 did not require fertilization.
In SAG-GO/2018, SAG-GO/2019 and
SAG-GO/2019–20 were needed to apply 270, 262.5 and, 260 kg ha-1 of
Triple superphosphate, respectively. However, 90 and 87.5 kg ha-1 of
potassium chloride were applied only in SAG-GO/2018 and SAG-GO/2019,
respectively.
The phytosanitary control was
performed after monitoring and evaluation of possible economic damage using
products registered for the common bean. For weed control, in the experiments
conducted in SAG-GO/2018, SAG-GO/2019, and SAG-GO/2019–20 seven days before
sowing (DBS) desiccation of the areas was done with the herbicide Paraquat - SL
200 g L-1 IA (2.0 L ha-1). In the experiment from
Formosa-GO/2018–19, ten DBS desiccation of the area was carried out with the
herbicides Aurora - EC 400 IA g L-1 IA (50 mL ha-1) and
Roundup - SL 445 IA g L-1 (2.0 L ha-1). In the experiment
from Cristalina-GO/2019, five DBS desiccation of the area was accomplished with
the herbicide Roundup - SL 445 IA g L-1 (2.0 L ha-1).
Table 1: Location, cropping season, Geographical coordinates, altitude, and previous crops of the experimental areas.
Location/cropping season |
Geographical
coordinates |
Altitude |
Previous crop |
|
Latitude (S) |
Longitude (W) |
m |
||
SAG-GO/2018 |
16º29`16.20” |
49º17`57.80” |
777 |
Corn |
Formosa-GO/2018-19 |
15º44`05.64” |
47º26`06.71” |
996 |
Corn |
Cristalina-GO/2019 |
16º56`14.70” |
47º46`02.70” |
905 |
Wheat |
SAG-GO/2019 |
16º29`15.22” |
49º17`54.74” |
776 |
Corn |
SAG-GO/2019-20 |
16º29`55.76” |
49º17`15.54” |
806 |
Corn |
SAG- Santo Antônio de Goiás
Table 2: Chemical characteristics of
the soil in the 0-0.20 m layer of the experimental areas where the field
experiments were conducted
Location/cropping season |
pH |
Ca |
Mg |
Al |
H+Al |
SB1 |
CEC2 |
BS3 |
SOM4 |
|||||
H2O |
mmolc dm-3 |
cmolc dm-3 |
% |
g kg-1 |
||||||||||
SAG-GO/2018 |
5.4 |
18.5 |
8.7 |
1 |
29 |
2.80 |
5.7 |
49 |
31.27 |
|||||
Formosa-GO/2018-19 |
5.6 |
45.6 |
12.0 |
0 |
24 |
6.57 |
9.0 |
73 |
46.93 |
|||||
Cristalina-GO/2019 |
5.8 |
38.5 |
13.6 |
0 |
22 |
5.69 |
7.9 |
72 |
51.90 |
|||||
SAG-GO/2019 |
5.8 |
17.0 |
9.8 |
0 |
24 |
2.87 |
5.3 |
54 |
28.91 |
|||||
SAG-GO/2019-20 |
5.8 |
18.2 |
9.0 |
0 |
24 |
3.09 |
5.5 |
56 |
23.00 |
|||||
Location/cropping season |
P |
K |
Cu |
Zn |
Fe |
Mn |
||||||||
mg dm-3 |
||||||||||||||
SAG-GO/2018 |
14.9 |
33 |
1.0 |
4.0 |
31.0 |
5.8 |
||||||||
Formosa-GO/2018-19 |
40.2 |
281 |
0.5 |
6.3 |
15.9 |
21.6 |
||||||||
Cristalina-GO/2019 |
45.7 |
187 |
2.1 |
8.1 |
32.7 |
22.2 |
||||||||
SAG-GO/2019 |
11.1 |
75 |
1.3 |
5.2 |
33.8 |
10.4 |
||||||||
SAG-GO/2019-20 |
24.9 |
145 |
1.3 |
5.2 |
11.6 |
40.8 |
||||||||
1SB = sum of bases; 2CEC = cation exchange capacity; 3BS
= bases saturation ((K + Ca + Mg)/Tcec) × 100, where Tcec = K + Ca + Mg + total
acidity at pH 7.0 (H + Al); 4SOM = Soil organic matter; 5SAG-
Santo Antônio de Goiás
Fig. 1: Rainfall, maximum and minimum mean temperatures during the experimental
periods. SAG-GO/2018 (A),
Formosa/2018-19 (B), Cristalina/2019
(C), SAG-GO/2019 (D) and SAG-GO/2019-20 (E)
Pre-emergence herbicide
application was done, between 2–3 days after sowing (DAS), in SAG-GO/2018, SAG-GO/2019, and SAG-GO/2019–20, using Gramoxone
- SL 200 IA g L-1 (2.0 L ha-1). Pre-emergence application
was also performed in Formosa-GO/2018–19 and Cristalina-GO/2019, using Gramocil
- SC 200 g L-1 IA (2.0 L ha-1). In SAG-GO/2018,
SAG-GO/2018–19, SAG-GO/2019 and Formosa-GO/2018–19, post-emergence herbicide
application was done, between 20 to 30 days after emergence (DAE), using Flex -
SL 250 g L-1 IA (1.0 L ha-1) and Fusilade - EW 250 g L-1
IA (0.75 L ha-1).
The experiments conducted in
SAG-GO/2018 and SAG-GO/2018–19
witnessed the occurrence of Bemisia
tabaci, requiring 2 applications of the insecticide Engeo Pleno - ZC 141 g
L-1 IA (125 mL ha-1). In the experiment from
Formosa-GO/2018–19 the insecticides Actara - WG 250 g kg-1 IA (200 g
ha-1), Benevia - OD 100 g L-1 IA (500 mL ha-1),
and Acephate - SP 750 g kg-1 IA (200 g ha-1) were used
for the control of B. tabaci with 3
applications in preventive and curative control. For the control of Etiella zinckenella, the biological Bt
insecticide (Bacillus thuringiensis)
was used in the experiment from Formosa-GO/2018–19.
For pathogen control in the
experiment from SAG-GO/2019 the fungicides Difere - SC 588 g L-1 IA (1.5 L ha-1), Fox - SC 150 g L-1
IA (400 mL ha-1) and Amistar Top - SC 200 g L-1 IA (400
mL ha-1) were used to control Phaeoisariopsis griseola, Colletotrichum
lindemuthianum and Erysiphe polygoni.
Sampling and evaluations to determine the nodule number (NN), nodule dry
weight (NDW), shoot dry weight (SDW), root dry weight (RDW), number of pods
(NP), number of grains (NG) and, grain yield (GY) were performed according to
Souza and Ferreira (2017).
The data obtained at the
different locations were subjected to group experiment analysis. In case of
significant differences between locations, the results of each location were
analyzed separately. On the analysis of variance was applied the F test (P ≤ 0.05) and, when Fc was significant, mean
values of the treatments were compared by Skott-Knott test (P ≤ 0.05). Statistical analyzes
were performes using the SISVAR software (Ferreira 2019).
Results
According to the group analysis,
locations showed significant differences among them. Thus, the data analysis
was performed separately by location.
Nodulation assessment
For most of the experiments, the
nodule number (NN) and nodule dry weight (NDW) were affected by the evaluated
treatments, and response variations were observed for each location. The
inoculation with only two doses of R. tropici (Rt) resulted in higher NN
in three of the five evaluated locations (Fig. 2), followed by the
co-inoculation treatments with two (Rt + Ab2l) and three (Rt + Ab3l) doses of A.
brasilense, which resulted in higher NN values in two of the five evaluated
locations (Fig. 2).
The absolute control (AC) showed
higher NN values than the registered commercial product (RP) in
SAG-2019 and SAG-2019/2020. The treatment with nitrogen fertilization (NfT)
resulted in NN values significantly lower than the other
treatments in SAG-2018, SAG-2019, and SAG-2019/20 (Fig. 2). In general, most of
the inoculated and/or co-inoculated treatments presented NN values
from 14 to 200 nodules plant-1 and NDW ranging from 17
to 190 mg plant-1. In this work, although no significant differences
were found in Cristalina-2019 for NN and NDW, co-inoculation with Rt+Ab1l
resulted in 19 nodules plant-1 and NDW of 19 mg plant-1,
providing a grain yield of 4457 kg ha-1, higher than the Rt
treatment.
NDW results were similar to NN,
showing significant differences between treatments in SAG-2018 and
SAG-2019/2020 (Fig. 2). The Rt + Ab2l treatment resulted in higher NDW values
in SAG-2018 and SAG-2019/2020. On the other hand, in SAG-2019 and SAG-2019/2020
higher NDW values were observed in the TA treatment (Fig. 2). Similar to what
was observed for NN, in SAG-2019 and SAG-2020, the TA treatment resulted in
higher NDW values compared to the PR treatment (Fig. 2). The TN treatment
behaved differently for NDW, and in SAG-2018 and SAG-2019 it resulted in the
lowest MNS values (Fig. 2).
Regarding NDW, significant
effect of treatments was found at three of the five evaluated locations. High
NDW values were found in treatments with co-inoculation, especially Rt + Ab2l,
Rt + Ab3l, and Rt + Ab4l co-inoculations in SAG-2019/2020, where these
treatments resulted in higher NDW values as compared to Rt and RP treatments
(Fig. 2).
Fig. 2: Effect of different doses of co-inoculation with R. tropici and A. brasilense applied at the phenological phase V2-V3
on the nodule number (A) and nodule
dry weight (B) of the common bean
cropped in different locations and sowing seasons. STA = SAG. AC = without co-inoculation and without N-fertilizer; NfT = 80 kg N ha-1
(20 kg N ha-1 applied at sowing and 60 kg N ha-1 applied
at V4 stage; Rt = seed inoculation with R. tropici (2.4x107
cells seed-1); Ab = spraying inoculation with A. brasilense in different concentrations (1l- 0.8×105
cells plant-1; 2l- 1.6×105 cells plant-1; 3l-
2.4×105 cells plant-1; and 4l- 3.2×105
cells plant-1); RP = Registered product (seed inoculation with R.
tropici-2.4x107 cells seed-1 and leaf inoculation
with A. brasilense- 2.4×105
cells seed-1). Means followed by the same letter, within each location,
do not differ by the Scott-Knott test (P <
0.05)
Fig. 3: Effect of different doses of co-inoculation with R. tropici and A. brasilense applied at the phenological phase V2-V3
on root dry weight (A) and shoot dry
weight (B) of the common bean
cropped in different locations and sowing seasons. STA
= SAG. AC = without co-inoculation and without N-fertilizer; NfT = 80 kg N ha-1
(20 kg N ha-1 applied at sowing and 60 kg N ha-1 applied
at V4 stage; Rt = seed inoculation with R. tropici (2.4x107
cells seed-1); Ab = spraying inoculation with A. brasilense in different concentrations (1l- 0.8×105
cells plant-1; 2l- 1.6×105 cells plant-1; 3l-
2.4×105 cells plant-1; and 4l- 3.2 × 105
cells plant-1); RP = Registered product (seed inoculation with R. tropici-2.4x107
cells seed-1 and leaf inoculation with A. brasilense- 2.4×105 cells seed-1). Means followed by the same letter, within each location,
do not differ by the Scott-Knott test (P <
0.05)
The Rt+Ab2l treatment provided
an increase in NDW in SAG-2019 and SAG-2019/2020, resulting in higher values
than the Rt treatment (Fig. 2). The NfT treatment was one of the
treatments with the lowest NN and NDW values in SAG-2018 and SAG-2019 (Fig. 2).
Assessment of growth parameters
For root dry weight (RDW),
significant differences were observed between treatments at all five locations.
In SAG-GO-2018, the highest values of RDW were found in the treatments Rt + Ab4l
and TN. The highest RDW values in Formosa-2018/2019 were found in the
treatments Rt + Ab3l, Rt + Ab4l, TA, and PR. While in Cristalina-2019 the
highest value of RDW was observed in the Rt treatment and, in SAG-2019 for the
Rt + Ab3l, Rt + Ab4l, and TA treatments. For SAG 2019/2020, the highest RDW
values were found in the treatments Rt + Ab2l, Rt, PR, and Rt + Ab4l (Fig. 3).
For shoot dry weight (SDW), statistical differences
were observed among treatments in four of the five evaluated locations. In
SAG-2018, higher SDW values were shown by TN and Rt + Ab4l treatments. In
Cristalina-2019, higher SDW values were shown by the treatments Rt, Rt + Ab2l,
TA, TN, Rt + Ab1l, PR and Rt + Abl4f and, in SAG-2019 the treatments Rt + Ab4l
and TA showed higher SDW values. In SAG-2019/2020, higher SDW values were found
in the treatments PR, Rt + Ab2l, Rt, and Rt + Ab4l (Fig. 3).
Yielding components and grain yield evaluation
For the number of pods (NP),
treatments were significantly differents in four of the five evaluated
locations. In SAG-GO-2018, higher NP values were shown by TN, Rt, PR, Rt + Ab1l,
Rt + Ab4l, and Rt + Ab2l treatments. In Cristalina-2019, higher NP values were
found in the treatments TA, Rt + Ab4l, and PR, and in SAG-GO-2019 in the
treatments PR, Rt + Ab4l, Rt + Ab3l, and Rt + Ab2l. For SAG-GO-2019/2020,
higher NP values were observed in PR and Rt treatments (Fig. 4). The PR and Rt +
Ab4l treatments significantly increased the NP in four of the five evaluated
locations (SAG-GO-2018, Cristalina-2019, SAG-2019 and SAG-2019/2020), with
higher values as compared to those of the treatments TN, Rt and TA (Fig. 4).
As for NP, significant
differences for number of grains (NG) were also observed between treatments in
four of the five evaluated locations. In SAG-GO-2018 higher NG values were
shown by TN, Rt, Rt + Ab1l, and Rt+Ab4l treatments. In Formosa-2018/2019,
higher NG values were observed in the Rt + Ab2l treatment. In Cristalina-2019,
higher NG values were provided by the treatments Rt + Ab4l, Rt + Ab2l and TA
and, in SAG-GO-2019/2020, by the treatments Rt, PR, Rt + Ab4l, TN, and Rt + Ab1l
(Fig. 4). In general, the Rt + Ab4l co-inoculation treatment resulted in NG
values higher than those of TN, PR, Rt, and TA treatments (Fig. 4).
Unlike the nodulation and growth
parameters, for the grain yield (GY) significant differences between treatments
were observed at all five locations. In SAG-2018, the treatments Rt + Ab4l, TN,
PR, Rt + Ab3l, TA, and Rt provided higher GY values. In Formosa-2018/2019,
higher GY values were noted in Rt + Ab1l and Rt + Ab4l treatments. In
Cristalina-2019 and SAG-2019, higher values of GY were found in the treatments
Rt + Ab1l and Rt + Ab3l and, in SAG-2019/2020 for the treatments Rt, PR and TA
(Fig. 4).
From the GY data, an assessment
of GY stability at the different locations was performed, whereby GY values
were plotted, within each site and for the average of the locations (Fig. 5),
ranging from light gray (worst GY) to dark gray (best GY).The treatment Rt + Ab3l
presented values of GY ranging from medium to high
Fig. 4: Effect of different doses of
co-inoculation with R. tropici and A. brasilense applied at phenological
phase V2–V3 on number of pods (A),
number of grains (B), and grain yield (C) of the common bean cropped in
different locations and sowing seasons. STA =
SAG. AC = without
co-inoculation and without N-fertilizer; NfT = 80 kg N ha-1 (20 kg N
ha-1 applied at sowing and 60 kg N ha-1 applied at V4
stage; Rt = seed inoculation with R. tropici (2.4 × 107 cells
seed-1); Ab = spraying inoculation with A. brasilense in different concentrations (1l- 0.8 × 105
cells plant-1; 2l- 1.6 × 105 cells plant-1;
3l- 2.4×105 cells plant-1; and 4l- 3.2 × 105
cells plant-1); RP = Registered product (seed inoculation with R.
tropici-2.4 × 107 cells seed-1 and leaf inoculation
with A. brasilense- 2.4 × 105
cells seed-1). Means followed by the same letter,
within each location, do not differ by the Scott-Knott test (P < 0.05)
(darker gray) in four of the
five evaluated locations, followed by the treatments Rt + Ab1l and Rt + Ab4l,
which presented GY values ranging from medium to high in three of the five
evaluated locations. Similarly, by the average of the five locations, it was
observed that the Rt + Ab3l treatment presented the best GY values (Fig. 5). By
the average of the five experiments, the treatment Rt + Ab3l increased the GY
in about 100, 93, 74 and 83 kg ha-1 as compared to the treatments
TN, PR, Rt, and TA, respectively (Fig. 5).
Discussion
As regards nodulation assessment is
concerned, treatments with simple inoculation with R. tropici and
co-inoculation with foliar application of different doses of A. brasilense
positively influenced nodulation by increasing the number of nodules and dry
mass of nodules plant-1. This occurs due to the positive synergism
of the two bacteria, in which A. brasilense is the first to colonize the
roots of plants and prepares them for the colonization of R. tropici, increasing
symbiotic efficiency. In this study, most inoculated and/or co-inoculated
treatments, NN values ranged from 14 to 200 nodules plant-1, while
NDW ranged from 17 to 190 mg plant-1.
Fig. 5: Grain yield (GY - kg ha-1) of common bean
co-inoculated with R. tropici and A. brasilense cultivated in different
seasons. Values highlighted in light gray (worst GY) and in dark gray (best
GY). 1AC = without co-inoculation and without N-fertilizer; NfT = 80
kg N ha-1 (20 kg N ha-1 applied at sowing and 60 kg N ha-1
applied at V4 stage; Rt = seed inoculation with R. tropici
(2.4 × 107 cells seed-1); Ab = spraying inoculation with A. brasilense in different
concentrations (1l- 0.8 × 105 cells plant-1; 2l- 1.6 × 105
cells plant-1; 3l- 2.4×105 cells plant-1;
and 4l- 3.2 × 105 cells plant-1); RP = Registered
product (seed inoculation with R. tropici-2.4 x 107 cells
seed-1 and leaf inoculation with A.
brasilense- 2.4 × 105 cells seed-1)
According to Oliveira and Santos (2011), well-nodulated
plants should have 15 to 30 nodules plant-1. A good indication of
symbiotic efficiency, which corresponds to good nodulation, are the plants that
present 100 to 200 mg of dry nodules in full bloom, are more likely to increase
the fixed N contents and, consequently, present high grain productivity
(Oliveira and Santos 2011). However, in the literature there are works in which
it presents high grain productivity with plants presenting low nodulation (NN
and NDW). Andraus et al. (2016), working with the inoculation of R.
tropici in the Estilo cultivar, reported NN of 16 nodules plant-1
and NDW of 39 mg plant-1, resulting in a grain yield of 3626 kg ha-1.
Indicating that, even with low nodulation, commercial bacteria can be efficient
and competitive with native rhizobia present in the soil.
In field experiments carried out by
Souza and Ferreira (2017), the inoculation of R. tropici (Rt) resulted
in NN and NDW values of 43 nodules plant-1 and 78 mg plant-1,
respectively, increasing grain yield in about 463 kg ha-1 compared
to the treatment without inoculation and without fertilization, indicating high
competitiveness of the SEMIA 4070 strain against native soil strains and high
efficiency of biological N fixation.
In this study, the co-inoculation of R.
tropici and A. brasilense provided better performance in nodulation
parameters, resulting in a greater number and mass of dry nodules,
corroborating the work of Souza and Ferreira (2017). Furthermore, the
beneficial effect of the co-inoculation of R. tropici and A.
brasilense on nodulation has been frequently observed in other crops, such
as soybean (Chibeba et al. 2015; Steiner et al. 2019) and peanut
(Silva et al. 2017; Steiner et al. 2019).
According to Steiner et al.
(2019), increase in nodulation driven by co-inoculation is due to the synergistic
effect produced by the two bacteria, in which A. brasilense qualifies
the root system so that it can be inhabited by R. tropici, improving the
nodulation rate, mainly in the root crown. Thus, beneficial results of the
association of symbiotic (R. tropici) and non-symbiotic (A.
brasilense) bacteria in legumes are mainly due to these rhizobacteria
having ability to fix N2, stimulate and produce growth
phytohormones, improve the activity of reductase enzyme activity, solubilize
soil phosphate, in addition to improving plant resistance to biotic and abiotic
stresses (Chibeba et al. 2015; Fukami et al. 2018a; Steiner et
al. 2019).
In this study, as well as several
available in the literature, we found that the use of nitrogen fertilizers
negatively affected plant nodulation, reducing the nodulation process (Sousa et
al. 2020), since the application of nitrogen fertilizers has negative
effects on the formation of nodules in legumes, through the inhibition of
phenolic compounds in plant metabolism, especially the synthesis and release of
isoflavonoids from legume roots (Steiner et al. 2019). A fact that the
energy expenditure by the plant is high in the process of biological N
fixation, unlike nitrogen fertilization, in which N is in the most accessible
form, which explains the reduction of nodulation when fertilizers are applied.
The results of co-inoculation with R.
tropici and A. brasilense applied via foliar route resulted in a
significant increase in root growth. This increase is associated with the main
mechanism of action of A. brasilense, the stimulation and production of
phytohormones in plants. This production of phytohormones with gibberellins and
auxins promotes a greater increase and development of lateral roots and root
hairs, consequently increasing the volume of exploited area in the soil and
improving plant performance (Chibeba et al. 2015; Vurukonda et al.
2016; Bulegon et al. 2017; Fukami et al. 2018b; Steiner et al.
2019). In addition, the application of A. brasilense to common bean improves
the performance of plants under water stress conditions, due to the increase in
the volume exploited by the roots (German et al. 2000). The use of
foliar application of A. brasilense, as well as in common bean, in soybean
significantly increased the length of the roots of the plants, in relation to
the co-inoculated plants (Puente et al. 2018).
Regarding the positive effect of
co-inoculation with four (Rt + Ab4l) and two (Rt + Ab2l) doses of A.
brasilense sprayed via foliar on the dry shoot mass, it is related to the
synergism between R. tropici and A. brasilense, through N
fixation, phytohormone production and phosphate solubilization and improved
resistance to biotic and abiotic stresses, resulting in greater growth of
healthier and more vigorous plants (Bulgarelli et al. 2013). However, in
the literature it is estimated that A. brasilense contributes from 5 to
18% of N fixation in legumes (Bremer et al. 1995). In addition,
co-inoculation with A. brasilense through synergism with R. tropici promotes
increased N fixation and plant growth (Filipini et al. 2021).
A fact that there are still few
studies with co-inoculation with the application of A. brasilense via
foliar spraying, as well as, in this study the benefits of this form of
application of co-inoculation with doses of A. brasilense promotes
greater development and performance of plants, with greater dry root mass and
dry shoots, increased branching, improved photosynthesis and water and nutrient
absorption, resulting in higher yields (Strzelczyk et al. 1994; Bashan et
al. 2004; Filipini et al. 2021).
Higher common bean yields are linked
to increases in the number of pods, number of grains per pod or grain weight
(Filipini et al. 2021). The N is the fundamental nutrient for most
crops, being directly related to the high rates, which determine the grain
weight in the filling period (Munier-Jolain et al. 2008; Filipini et
al. 2021). In this study, in most of the locations considered, the
co-inoculation with the foliar spraying of four doses of A. brasilense
(Rt + Ab4l), increased the number of pods plant-1, which presented
the same effect and superior to the treatment with nitrogen fertilization
(NfT). The results for NP obtained in our work were superior to those found in
the works of Steiner et al. (2019) and Tocheto and Boiago (2019).
According to Steiner et al. (2019) the explanation for the increase in
the number of grains is associated with the combination of R. tropici
and A. brasilense, which stimulates flower opening and pod formation,
fundamental factors for achieving high productivity in common bean. Likewise,
Peres et al. (2016) also reported that a combination of R. tropici
and A. brasilense significantly influences the NP.
Furthermore, the NG values found in
this study were higher than the values reported by Steiner et al.
(2019). According to Tocheto and Boiago (2019), the co-inoculation of R.
tropici and A. brasilense directly influences the number of grains
and grain production of common bean. The effect may be related to the
environmental conditions associated with the potential of each genetic material
for co-inoculation (Ferri et al. 2017; Braccini et al. 2016;
Filipini et al. 2021), mainly by A. brasilense applied via the leaf, which this is subject to
different climate conditions in the interaction between bacteria and plants in
the phyllosphere, which may affect the potential of the technique, which did
not happen in this study. According to Carvalho et al. (2018)
performance of the combination of Rhizobium and Azospirillum
depends on some factors, such as the native microbiota, plant species and
varieties, as well as other edaphoclimatic conditions.
Co-inoculation treatment with foliar
spraying applying three doses of A. brasilense (Rt + Ab3l) provided
greater GY than treatment with single inoculation with R. tropici (Rt) (Fig.
2), as well as, in studies carried out by Hungria et al. (2013), Souza
and Ferreira (2017) and Bettiol et al. (2021). In the work carried out
by Schossler et al. (2016), the co-inoculation of R. tropici and A.
brasilense led to a GY of 2448.45 kg ha-1, increasing the GY by
13.5% (292 kg ha-1) in relation to the single inoculation with A.
brasilense and 5.7% (131 kg ha-1) for R. tropici. Bettiol
et al. (2021) reported identical results in their study, in which the
co-inoculation of R. tropici and A. brasilense provided a
significant increase in GY of 403, 188, 392 and 363 kg ha-1 compared
to treatments inoculated with R. tropici, N-fertilization with 90 kg N
ha-1, N-fertilization with 45 kg N ha-1 and control
treatment without fertilization and co-inoculation, respectively.
Our work demonstrates the potential of
this technology from the positive results of co-inoculation, with the combined
use of R. tropici and foliar application of A. brasilense, which
results in greater yield and grain production in the common bean crop. This
technology can bring economic advantages and reduce environmental impacts under
field conditions in commercial common bean production farms. This technology
can be applied not only locally, but regionally and nationally in countries
that have similar edaphlocimatic conditions for common bean cultivation, being
a great alternative for increasing common bean yield in developing countries, where
nitrogen fertilization is inaccessible, due to
the high cost in the production system.
The results indicate that the co-inoculation technique
with R. tropici applied to the seed and, associated with A.
brasilense applied via foliar spraying, is an alternative for partial or
total replacement of the use of nitrogen fertilization in the common bean crop.
Furthermore, co-inoculation with two doses of Rhizobium and three doses
of Azospirillum via foliar spray resulted in increased nodulation,
significant increase in shoot and root biomass, increased number of pods and
grains, and increased and greater stability of grain yield, when compared to
nitrogen fertilization, registered product, simple inoculation with R.
tropici and without fertilization and without co-inoculation.
Conflict of Interest
The authors declare no conflict of interest
Data Availability
The reported data can be made available
upon requesting to the corresponding author
Ethics Approval
Not applicable in this research work
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