Interference of Herbicides in Association of Diazotrophic
Bacterium Nitrospirillum amazonense and Sugarcane Pre-Sprouted Seedlings
Luana Carolina Gomes
Jonck1,2, Márcia Maria Rosa Magri1 and Patricia Andrea
Monquero1*
1FAPESP Scholarship Process 2020/03715-4
2Center for Agricultural Sciences, Federal University of São Carlos,
Araras, São Paulo, Brazil
*For correspondence:
pamonque@ufscar.br
Received 24 November 2021; Accepted 20 January
2022; Published 30 March 2022
Abstract
Microbial inoculant containing
cells of Nitrospirillum amazonense
is a recent technology that has been used in association with pre-sprouted
seedlings to sustainably increase the productivity of sugarcane. This study
aimed to assess the sensitivity of the rhizobacterium N. amazonense
to the herbicides imazapic and indaziflam
and the effect of this inoculation and herbicide treatments on sugarcane
pre-sprouted seedlings. The In vitro sensitivity of the N. amazonense to the herbicides was assessed using the
minimum inhibitory concentration technique (first assay). In this research, we
evaluated imazapic (200 g a.i.
ha-1) and indaziflam (100 g a.i. ha-1) at five doses: recommended dose
(1×D), twice the recommended dose (2×D), one and a half of the recommended dose (1.5×D), half the
recommended dose (0.5×CD), a quarter of the recommended dose (0.25×CD) and
control treatment. The sensitivity of N. amazonense
to imazapic and indaziflam
applied at commercial doses on autoclaved soil was assessed in the second
assay. The bacterial population count was performed using the most probable
number technique (McCrady Table). The third assay assessed five herbicide
treatments (clomazone (720 g a.i. ha−1),
imazapic (200 g a.i. ha−1),
tebuthiuron (800 g a.i. ha−1),
indaziflam (75 g a.i. ha−1),
sulfentrazone (800 g a.i.
ha−1) and control without herbicide) applied in pre-planting
of pre-sprouted seedlings of the variety RB 966928 in the presence and absence
of the inoculant N. amazonense. The results
showed that the presence of indaziflam did not
interfere with the In vitro growth of the bacterium N. amazonense, regardless of the dose. Imazapic
caused significant inhibition of bacterial In vitro growth from the
recommended dose (200 g a.i. ha-1). The N. amazonense count in the soil of treatments that
received indaziflam and imazapic
application did not differ compared to the soil without herbicide. The
pre-sprouted seedlings of the variety RB966928 showed high sensitivity to the
herbicide imazapic, regardless of N. amazonense inoculation. Clomazone, tebuthiuron,
and sulfentrazone did not interfere with the
growth-promoting effect of N. amazonense. The
results showed that the recommended dose of the herbicides tested does not
impair the growth promoting effect of N. amazonense,
and the inoculation of the pre-sprouted seedlings does not alter their
sensitivity to herbicides, although the selectivity of the seedlings is
differential among herbicides. Therefore, it may be concluded that the combined
use of these technologies is a viable alternative to increase sugarcane
productivity in a more sustainable way.
© 2022 Friends Science Publishers
Keywords: Pre-sprouted seedlings; Plant growth-promoting bacteria; Intoxication; Herbicides
Introduction
The growing demand in the
sugar-energy sector has led to the search for new sugarcane production
technologies aimed at increasing raw material productivity and quality. In this
sense, the multiplication system through pre-sprouted seedlings (PSS) and the use
of inoculants based on plant growth-promoting bacteria are among the
technological innovations employed in the sector (Pereira et al. 2013;
Garcia 2016).
The PSS technology is a multiplication system used to implement
previously treated seedlings in the plantation, providing high phytosanitary
quality to the sugarcane field, high clonal standard, homogeneity, and vigor,
and reduction in the volume of plant material used in the planting process
(Ventura 2017).
On the other hand, plant growth-promoting bacteria (PGPB) consists of a
group of microorganisms with the ability to associate with plants and stimulate
their growth (Oliveira et al. 2003). The PGPB mechanisms differ between
species and can benefit plant growth directly through phytohormone production,
phosphate solubilization, and biological nitrogen fixation, or indirectly
through siderophore production and induction of resistance systemic to pathogens
(Costa et al. 2014). PGPB can promote an increase in the bud sprouting
rate of associated plants and the rapid establishment of lateral and
adventitious roots through phytohormone production, resulting in the
exploration of a higher soil volume and, consequently, better water and
nutrient absorption (Lopes 2013).
Sugarcane can associate with a large number of species of plant
growth-promoting bacteria. Thus, the inoculation of these microorganisms in the
crop has become a viable alternative to the sugar-energy sector to increase
sustainably of the raw material productivity and quality, reducing costs and
environmental impacts (Ferreira et al. 2018). Studies applied in the
area have led to the development of a liquid microbiological inoculant from the
bacterium Nitrospirillum amazonense specific for sugarcane cultivation. The
product can generate sugarcane productivity gains of up to 18% (Embrapa 2018).
The presence of agrochemicals can compromise the efficiency of PGPB
performance, as the contact with these molecules can cause specific damage to
bacterial cells, such as inhibition of protein synthesis, DNA alterations, and
oxidative destruction of membranes, leading to bactericidal and bacteriostatic
effects or even harming the biological nitrogen fixation effectiveness of these
microorganisms (Procópio et al. 2013; Lino
2018).
Studies with several herbicides commonly used in sugarcane have shown
their toxic effect on PGPB development, such as the products imazapyr, ametryne, and oxyfluorfen (Procópio
et al. 2014), paraquat, amicarbazone,
clomazone, diuron, metribuzin, 2,4-D (Procópio et
al. 2013), and isoxaflutole (Silva et al.
2014).
In contrast, Pies et al. (2017) observed beneficial effects of
the application of diuron, imazapic, and clomazone on
the development of the diazotrophic bacterium Burkholderia
tropica. In this case, these herbicides acted to
stimulate the microorganism development, which can be explained by the ability
of some bacteria to degrade herbicide molecules, using their chemical compound
as a source of energy and carbon.
Das and Debnath (2006) also reported stimulant effects of herbicides on
diazotrophic microorganisms, as the presence of the herbicide oxyfluorfen led
to an increase in microbial activity, resulting in higher atmospheric nitrogen
fixation and phosphate solubilization by these microorganisms.
Pre-sprouted seedlings are more sensitive to soil residues because they
are transplanted with a root system already formed, which may cause
intoxication by herbicides applied in pre-and post-planting (Silva et al. 2018).
PGPB inoculation stimulates the rapid growth of lateral and adventitious roots
(Chaves et al. 2015), which may cause increased sensitivity of the
seedlings to phytotoxicity by herbicides present in the soil.
Thus, studies on the compatibility of herbicides to PGPB associated with
the sugarcane and the herbicide interference in the sensitivity of sugarcane
pre-sprouted seedlings inoculated with these microorganisms are required. This
study aimed to assess the compatibility of the herbicides imazapic
and indaziflam with the bacterium N. amazonense and the sensitivity of inoculated
pre-sprouted seedlings for the application of the herbicides clomazone, imazapic, tebuthiuron, indaziflam, and sulfentrazone.
Materials and Methods
In vitro assays of herbicide compatibility with the bacterium N. amazonense
Minimum inhibitory concentration assessment: The experiment was carried out
at the Laboratory of Agricultural and Molecular Microbiology (LAMAM) of the
Center for Agricultural Sciences at UFSCar, Araras, SP, Brazil.
The tests were carried out using
the strain of N. amazonense (BR 11145)
obtained from the Diazotrophic Bacteria Collection at Embrapa
Agrobiology. In inoculant preparation, the cells of the bacterium N. amazonense were activated and multiplied in 200 mL of
nutrient broth (NB), whose formulation, in g/L of distilled water, consisted
of: 1.0 (meat extract), 2.0 (yeast extract), 5.0 (peptone), and 5.0 (sodium
chloride). The culture was incubated in a shaker at 30°C and 150 rpm until the
medium became cloudy, reaching an optical density (OD600nm) of
approximately 0.8.
The experimental design was completely
randomized in a 2 × 5 factorial scheme, consisting of two herbicides and five
doses, with three replications. Two herbicides registered for sugarcane, imazapic (recommended dose – 200 g a.i.
ha-1) and indaziflam (recommended dose –100
g a.i. ha−1) were tested at five
doses (recommended dose (1 × D), twice the recommended dose (2 × D), one and a half of the
recommended dose (1.5 × D), half the recommended dose (0.5 × D), a quarter of
the recommended dose (0.25 × D) and control treatment, each dose being
considered a treatment.
In the preparation of herbicide
solutions, the herbicides were submitted to serial dilutions to obtain the
concentrations that represented the previously established doses. Subsequently,
they were filtered on a membrane with 0.2-micrometer pores for sterilization.
The methodology adopted to
assess the minimum inhibitory concentration was based on that described by Procópio et al. (2011). Therefore, the herbicide
solutions were mixed in a 125 mL Erlenmeyer flask with 50 mL of NB. The control
treatments received the same volumes of sterile distilled water. Finally, 0.1
mL of the microbial inoculant was added to the medium.
The treatments were incubated in
a shaker at 30 ± 2°C and 150 rpm for 48 h. The N. amazonense
cells were quantified by absorbance in a spectrophotometer (600 nm) through the
correlation in a standard curve from a pre-culture of a pure sample in NB,
according to the methodology based on Silva et al. (2008).
Evaluation of N. amazonense resistance to soil herbicide application: The soil used in this experiment was collected from a native Table 1:
Chemical analysis of soil samples used in the experiment
Latossolo Vermelho Escuro (Oxisol) |
|||||||||
P |
OM |
pH |
K |
Ca |
Mg |
H+Al |
SB |
CEC |
V |
mg/dm3 |
g/dm3 |
CaCI2 |
mmolc/dm3 |
|
|
% |
|
|
% |
19 |
32 |
5.4 |
2.7 |
60 |
10 |
31 |
72.7 |
103.7 |
70 |
*pH measured in CaCl2 0.01
M solution
Table 2: Composition of the LGI medium
Reagent |
Quantity/L |
Granulated sugar |
5 g |
Agar |
1.4 g/L |
0.5% bromothymol blue in 0.2 N KOH |
5 mL |
1% w/v calcium chloride dihydrate |
2 mL |
1% w/v ferric chloride hexahydrate |
1 mL |
Yeast extract |
0.02 g/L |
10% w/v dibasic potassium phosphate |
2 mL |
10% w/v monobasic potassium phosphate |
6 mL |
0.1% w/v sodium molybdate dihydrate |
2 mL |
Potassium nitrate |
1 g/L |
10% w/v magnesium sulfate heptahydrate |
2 mL |
Source: Adapted from Döbereiner et al. (1999)
forest, located at the Center
for Agricultural Sciences (CCA–UFSCar), Araras, SP, Brazil, at a depth of 0.10 m, without previous
pesticide application. The soil chemical analysis was carried out by the
Laboratory of Soil Chemistry and Fertility of the CCA/UFSCar
(Table 1).
Soil samples (1000 g) were crushed, sieved through a 2-mm mesh,
homogenized, and subjected to the tyndallization
process, which consists of soil sterilization to eliminate microorganisms (Basseto et al.
2008). Therefore, the soil was placed under steam pressure from an autoclave
for 20 minutes for three consecutive days, according to the methodology
described by Hungria and Araújo (1994).
The experimental design in this assay was completely randomized with two
herbicides in the presence and absence of the bacterium N. amazonense, with four replications.
Control treatments, one without herbicide and inoculant and another with only
inoculant, were also assessed. The doses for the herbicides imazapic
and indaziflam were 200 and 100 g a.i.
ha−1, respectively. Before being applied to the sterile soil,
the herbicides were serially diluted to obtain the established doses and
previously filtered on 0.22 µm
membranes to sterilize the solution.
The inoculant with N. amazonense cells
was prepared as described in the firsty assay. The
soil microbial inoculation was carried out with the inoculant application at a
dose equivalent to 1.5 L ha−1. The same volumes of sterile
distilled water were applied for the control treatments. The soil samples were
incubated at room temperature for 48 h.
The methodology used for quantifying N. amazonense
cells was based on that proposed by Videira et al.
(2007). After the incubation period, 10 g of soil were collected from each
treatment, being diluted in 90 mL of saline solution and then serially diluted
by adding 1 mL of the original dilution into test tubes with 9 mL of saline
solution. This process was repeated until the 10−6 dilution. A
sample (in triplicate) of 0.1 mL from each dilution was inoculated into flasks
with 5 mL of semi-solid LGI culture medium (Table 2), which is a semi-selective
medium for N. amazonense isolation.
Subsequently, the inoculated flasks were incubated at 30°C for 7 days.
The bacterial population count was performed using the most probable
number (MPN) technique, using the McCrady Table for three replicates of each
dilution. Bacterial growth was detected by visualizing the formation of a
characteristic veil-shaped surface film on the semi-solid medium.
Sensitivity of sugarcane
pre-sprouted seedlings inoculated with N. amazonense
to herbicide application
The experiment was carried out
in a greenhouse and the experimental units consisted of polyethylene pots with
a 6.0-L volumetric capacity filled with soil samples classified as Latossolo Vermelho distrófico (Oxisol), whose physico-chemical
analysis was carried out by the Laboratory of Soil Chemistry and Fertility of
the CCA/UFSCar (Table 1).
Sugarcane planting was carried out using pre-sprouted seedlings (PSS)
with the technology AgMusa. The seedlings of the
variety RB966928 were planted 60 days after bud sprouting. This variety has
characteristics including high tillering, medium useful period of
industrialization, and early to medium maturation. The variety stands out as
the most planted in the state of São Paulo (Ridesa
2020). The seedlings were irrigated by a sprinkler system, according to the
evapotranspiration demand.
The experimental design for pre-planting application was completely
randomized with five replications in a 6 × 2 factorial scheme. The first factor
consisted of the application of the herbicides clomazone (720 g a.i. ha−1), imazapic
(200 g a.i. ha−1), tebuthiuron (800 g a.i. ha−1),
indaziflam (75 g a.i. ha−1),
and sulfentrazone (800 g a.i.
ha−1), in addition to the non-herbicide application (control
treatment). The herbicide treatments were applied with a CO2-pressurized
knapsack sprayer set at a constant pressure of 245.16 kPa and a boom equipped
with four flat fan spray tips (110.03). The spray solution volume was 200 L ha−1.
The second factor was (i) presence, and (ii) absence
of the microbial inoculant.
The methodology used for N. amazonense
inoculation was based on Reis and Urquiaga (2009), being carried out by immersing the
seedling root system in a solution with a concentration of 1 × 10−8
CFU mL−1. Seedling transplanting was carried out immediately
after the product inoculation.
The assessments were carried out at 7, 14, 28, and 56 days after
herbicide application (DAA). Visual assessments of herbicide toxicity were
carried out in a range between 0 (absence of symptoms) and 100 (plant death),
according to the methodology proposed by Velini et
al. (1995).
The sugarcane plants were
assessed at 56 DAA and their height (cm) was determined considering the
distance from the base to the first leaf insertion, while the leaf area (cm2)
was obtained using an LI-COR LI-3000C portable leaf area meter. Then, the
plants were cut close to the ground and the shoot dry biomass (g) was
determined in an oven at 65°C for 48 h. The pots were disassembled, and the
roots were Table 3: Log10 of the most probable number of CFU of N. amazonense per gram of soil
Treatment |
g a.i.
ha-1 + L ha-1 |
log MPN CFU g-1
of soil |
Imazapic + N. amazonense |
200 + 1.5 |
5.82 a |
Indaziflam + N. amazonense |
100 + 1.5 |
5.37 a |
N. amazonense |
0 + 1.5 |
5.28 a |
Control |
- |
0.00 b |
LSD |
0.63 |
|
CV% |
7.25 |
*Means followed by the same
letter do not differ statistically from each other by Tukey’s test at the 5%
probability
Fig. 1: Absorbance of N. amazonense in a
medium with different doses of the herbicides imazapic
and indaziflam
washed and dried in an air-circulation oven at 65°C for 48 h to
determine the length (cm) and root dry biomass (g).
Statistical analysis
The minimum inhibitory concentration
assessment data were subjected to analysis of variance and regression curves
were constructed using the SIGMA-Plot when significant. The results obtained
from the McCrady Table were subjected to logarithmic transformation and later
submitted to analysis of variance. In the second and thirdy
assay the means were compared by Tukey’s test at the 5% probability when the
analysis of variance was significant.
Results
In vitro compatibility between the herbicides imazapic
and indaziflam and the diazotrophic bacterium N. amazonense
The results of absorbance
regarding the growth of N. amazonense cells in
the medium with different imazapic doses (Fig. 1)
showed a negative interference of the herbicide on the microbial growth from
the commercial dose. The N. amazonense growth
in medium with the herbicide indaziflam did not
differ from the control treatment (Fig. 1) for all tested doses (0.25 × D, 0.5 ×
D, 1 × D, 1.5 × D and 2 × D), showing herbicide selectivity.
Sensitivity of N. amazonense to soil-applied herbicides
The sensitivity of the bacterium
N. amazonense to the herbicides imazapic and indaziflam applied
to the soil is shown in Table 3. Fig. 2 shows the bacterial growth through the
formation of a typical film and change in the culture medium color. The growth
of the bacterium N. amazonense occurred in the
presence of both herbicides. No significant differences were observed in the
MPN of CFU g−1 of soil between the treatments that received
the herbicides imazapic and indaziflam
relative to the treatment that received only inoculant (Table 3).
Sensitivity of sugarcane
pre-sprouted seedlings inoculated with N. amazonense
to herbicide application
A significant interaction was
observed between herbicides and the inoculant applied to sugarcane pre-sprouted
seedlings (Table 4). The assessment carried out at 7 DAA, considering
non-inoculated seedlings, showed that the herbicides did not differ from each
other regarding phytotoxicity, with low values. However, the herbicide indaziflam promoted 41% phytotoxicity with the previous
seedling inoculation, with 0% in the treatment without inoculation. The other
herbicides did not differ from the control, regardless of the presence or
absence of the inoculant (Table 4).
Table 4: Percentage of phytotoxicity of herbicides applied in the pre-planting
of sugarcane pre-sprouted seedlings of the variety RB966928 with and without N.
amazonense inoculation assessed at 7, 14, 28, and
56 DAA
Treatment |
|
7 DAA |
14 DAA |
28 DAA |
56 DAA |
||||
|
g a.i. ha−1 |
I |
NI |
I |
NI |
I |
NI |
I |
NI |
indaziflam |
75 |
0.00 aB |
41.00 aA |
20.00 bcB |
45.00 aA |
50.00 abA |
50.00 aA |
70.00 bA |
56.00 bA |
imazapic |
200 |
10.00 aA |
5.00 bA |
62.00 aA |
40.00 aB |
64.00 aA |
53.00 aA |
97.00 aA |
88.00 aA |
clomazone |
720 |
11.00 aA |
5.00 bA |
54.00 aA |
33.00 abB |
45.00 abA |
50.00 aA |
32.00 cA |
26.00 cA |
tebuthiuron |
800 |
0.00 aA |
0.00 bA |
1.00 cA |
11.00 bcA |
0.00 cA |
0.00 bA |
0.00 dA |
0.00 dA |
sulfentrazone |
800 |
5.00 aA |
3.00 bA |
38.00 abA |
46.00 aA |
39.00 bB |
55.00 aA |
16.00 cdB |
36.00 cA |
Control |
– |
0.00 aA |
0.00 bA |
0.00 cA
|
0.00 cA |
0.00 cA |
0.00 bA |
0.00 dA |
0.00 dA |
LSD |
|
15.69 |
5.87 |
20.45 |
8.49 |
17.64 |
6.22 |
13.10 |
6.18 |
CV (%) |
|
167.48 |
165.17 |
49.86 |
54.62 |
37.08 |
34.50 |
26.57 |
33.07 |
*I = inoculated.; NI = non-inoculated. Means followed
by the same lowercase letters in the column and uppercase letters in the row do
not differ statistically from each other by Tukey’s test at the 5% probability
Table 5: Height, leaf area, and shoot biomass of sugarcane pre-sprouted
seedlings of the variety RB966928 with and without inoculation of N. amazonense assessed at 56 DAA
Treatment |
g a.i. ha−1 |
Height (cm) |
Leaf area (cm2) |
Biomass (g) |
|||
|
|
I |
NI |
I |
NI |
I |
NI |
indaziflam |
75 |
13.00 bA |
14.00 bcA |
40.61 cA |
90.33 bcA |
4.42 bcA |
2.97 abA |
imazapic |
200 |
7.60 bA |
11.40 cA |
8.92 cA |
35.46 cA |
1.64 cA |
1.48 bA |
clomazone |
720 |
25.20 aA |
21.20 abA |
412.08 abA |
257.70 aB |
7.38 aA |
4.28 abB |
tebuthiuron |
800 |
27.00 aA |
21.80 aA |
408.30 abA |
257.01 aB |
7.18 abA |
4.18 abB |
sulfentrazone |
800 |
25.40 aA |
18.20 abcB |
342.90 bA |
183.89 abB |
6.93 abA |
4.06 abB |
control |
– |
31.00 aA |
22.40 aB |
469.75aA |
276.42 aB |
8.40 aA |
5.26 aB |
LSD |
|
5.37 |
2.30 |
82.84 |
34.54 |
2.33 |
0.68 |
CV (%) |
|
19.24 |
21.71 |
25.41 |
27.94 |
34.24 |
26.38 |
*I = inoculated.; NI =
non-inoculated. Means followed by the same lowercase letters in the column and
uppercase letters in the row do not differ statistically from each other by
Tukey’s test at the 5% probability, within each biometric parameter
The assessment carried out at 14 DAA showed that only tebuthiuron did not cause phytotoxicity in the seedlings
with or without inoculation. A distinct response was found between herbicides
regarding the effect of inoculation. In this sense, the inoculated seedlings
continued with higher phytointoxication caused by indaziflam compared to the non-inoculated seedlings.
However, the opposite occurred for imazapic and
clomazone, with injuries corresponding to 62 and 54% in inoculated seedlings
and 40 and 33% in non-inoculated seedlings, respectively.
The herbicide sulfentrazone showed no
influence of microbial inoculation in PSS on the phytotoxicity assessed at 14
DAA. However, the assessments carried out at 28 and 56 DAA showed a higher recovery
of plants inoculated with rhizobacteria, with final phytotoxicity of 16% in
inoculated plants and 36% in non-inoculated plants. This result shows that
seedling inoculation using rhizobacteria may assist in reducing the
phytotoxicity of some herbicides.
The herbicides indaziflam, imazapic,
clomazone, and tebuthiuron showed no statistical
differences between inoculated and non-inoculated plants at the final
assessments (28 and 56 DAA). Imazapic showed the
highest phytotoxicity. Phytotoxicity values of 97 and 88% were observed at 56
DAA for seedlings with and without inoculation, respectively.
The herbicide indaziflam showed lower
phytotoxicity than that promoted by imazapic but
still considered high (70 and 56% for inoculated or non-inoculated seedlings,
respectively).
The herbicides clomazone (720 g a.i. ha−1)
and sulfentrazone (800 g a.i.
ha−1) showing values ranging from 16 to 36% of phytotoxicity. Tebuthiuron (800 g a.i. ha−1)
was the most selective herbicide in the study, not differing from the control
without application.
Seedling height showed an interaction between herbicides and inoculation
of the rhizobacterium N. amazonense (Table 5).
The herbicide sulfentrazone and the control without
application showed significant differences regarding the inoculation factor,
with a favorable inoculation effect.
As observed for height, the herbicides imazapic
and indaziflam also stood out negatively for leaf
area and shoot dry biomass, with lower means compared to the control without
application (Table 5). These herbicides showed no significant differences
between the seedlings that were or were not previously inoculated for the
assessed parameters.
The inoculation of pre-sprouted
seedlings promoted increases above 40% in leaf area and shoot dry biomass compared
to non-inoculated seedlings for clomazone, tebuthiuron,
sulfentrazone, and control.
The analysis of seedling growth variables allows us to state that the
application of the herbicides clomazone (720 g a.i.
ha−1), tebuthiuron (800 g a.i. ha−1), and sulfentrazone
(800 g a.i. ha−1) does not interfere
with the growth-promoting effect of the bacterium, considering that the
seedlings showed a favorable effect of inoculation by N. amazonense
even in the presence of these herbicides. The inoculation of seedlings also did
not increase their sensitivity to herbicide treatments applied in the
pre-planting of pre-sprouted seedlings. Thus, the use of inoculation is
suitable in this planting system.
Table 6: Root length and root dry biomass of sugarcane pre-sprouted seedlings of
the variety RB966928 transplanted with and without inoculation of N. amazonense assessed at 56 DAA
Treatment |
|
Root length (cm) |
Root biomass (g) |
||
|
g a.i. ha−1 |
I |
NI |
I |
NI |
indaziflam |
75 |
13.00 cA |
20.90 bA |
10.94 bA |
9.43 bA |
imazapic |
200 |
7.00 cA |
7.00 bA |
4.66 cA |
5.23 bA |
clomazone |
720 |
65.50 abA |
64.40 aA |
18.13 aA |
9.05 bB |
tebuthiuron |
800 |
56.80 bA |
68.80 aA |
12.76 abA |
9.70 bA |
sulfentrazone |
800 |
54.80 bA |
59.80 aA |
11.52 bA |
8.12 bA |
control |
– |
73.20 aA |
65.20 aA |
17.17 aA |
15.15 aA |
LSD |
|
10.74 |
4.98 |
4.12 |
1.49 |
CV (%) |
|
16.48 |
20.14 |
26.69 |
25.52 |
*I = inoculated.; NI =
non-inoculated. Means followed by the same lowercase letters in the column and
uppercase letters in the row do not differ statistically from each other by
Tukey’s test at the 5% probability
Fig. 2: Surface film formation and color change (Tube b) confirm the growth of N.
amazonense on the semi-solid LGI culture medium. Araras 2021
Length and root dry biomass showed no inoculation effect, except for the
treatment with clomazone (720 g a.i. ha-1),
which showed higher root dry biomass in inoculated seedlings (Table 6).
Discussion
The results regarding in vitro
compatibility between the herbicides imazapic and indaziflam and the diazotrophic bacterium N. amazonense showed that bacterial growth was inhibited
by the herbicide imazapic from the recommended dose.
Whereas herbicide indaziflam did not interfere with
the In vitro growth of N. amazonense at
the doses evaluated. Schwerz et al. (2017a)
also observed no effect of imazapic on the Azospirillum amazonense growth when it was grown in a medium with imazapic at the commercial dose under In vitro
conditions. The selectivity of imazapic at commercial
doses has been observed over other species of diazotrophic bacteria. Procópio
et al. (2014) also found no toxic effect of this herbicide on Herbaspirillum seropedicae
growth. Also, other studies have found no changes in the growth and biological
nitrogen fixation activity of bacterial cells of Gluconacetobacter
diazotrophicus when grown in a medium with the
herbicide imazapic (Procópio
et al. 2011, 2013).
Imazapic is an
acetolactate synthase (ALS)-inhibiting herbicide. The ALS enzyme participates
in the biosynthesis of the amino acids valine, leucine, and isoleucine in
microorganisms and plants (Christofoletti 2001). The
highest interference of herbicides on the soil microbiota occurs when they act
on the biosynthesis of amino acids or metabolic pathways common to
microorganisms and plants (Santos et al. 2006).
Imazapic showed
selectivity to N. amazonense up to the
recommended dose but considering literature reports confirming its selectivity
over other species of diazotrophic bacteria, we cannot infer that the
selectivity occurs due to a resistance of these microorganisms to the mechanism
of action of the herbicide. Procópio et al.
(2014) observed selectivity of imazapic on the
bacterium H. seropedicae, but a significant
inhibition in the growth of the microorganisms was found when imazapyr, an
herbicide belonging to the same mechanism of action and chemical group, was
assessed.
The selectivity of an herbicide is not only associated with the active
ingredient, mechanism of action, or chemical group but also with factors
related to the physico-chemical characteristics of
the commercial product. Compounds present in agro-chemical
formulations, such as solvents, surfactants, and wetting agents, may be
directly associated with the toxic herbicide effect on microorganisms (Santos et
al. 2004).
The increased herbicide dose potentiated its negative effect on strain
growth. Childs (2007) observed in In vitro tests that herbicides are
potentially toxic to microorganisms at high concentrations, with frequent
inhibitory effects on the quantity and activity of these organisms. However,
the contact between the herbicide and microorganisms in In vitro tests
is theoretically higher than under field conditions.
On the contrary, Schwerz et al. (2017a)
observed no negative interferences in the growth of the bacterium A. amazonense with an increase in the dose of the
herbicide imazapic since no change was observed in
the medium turbidity compared to the control treatment when the product was
applied at the highest concentration. Similarly, Pies et al. (2017)
verified that the growth of the diazotrophic bacterium Burkholderia
tropica in a medium with different imazapic doses, instead of reducing, had an increase in
optical density values with increasing herbicide doses.
Tironi et
al. (2009) emphasized that microorganisms are subjected to maximum exposure
to toxic herbicide molecules in In vitro tests, which does not occur
under field conditions, where external factors act on the chemical, reducing
its toxicity. Thus, herbicides identified as selective to microorganisms in
laboratory tests are likely to present selectivity under field conditions.
Similar to our results regarding sensitivity of N. amazonense
to soil-applied herbicides, Koçak et al.
(2021) found no negative or positive effects of the herbicide indaziflam applied to the soil on the microbial population.
Torres et al. (2018) found that the application of the herbicide indaziflam did not cause damage to soil microorganisms,
increasing the microbial population. Several studies in the literature have
reported the absence of negative effects of the herbicide imazapic
on species of diazotrophic bacteria associated with sugarcane (Procópio et al. 2011, 2013, 2014; Pies et al.
2017; Schwerz et al. 2017b).
Pesticide application can positively affect the soil microbiota when the
molecules are likely to be metabolized by microorganisms, or negatively
interfere with it when they intoxicate the microbial population (Ferreira
2016). Several strategies can be used by microorganisms to metabolize
herbicides: (a) catabolism: the herbicide molecule is absorbed and broken down,
generating energy; and (b) cometabolism: the
herbicide is transformed by metabolic reactions, but it is not used as an
energy source (Childs 2007).
In the catabolism process, microorganisms use energy from herbicide
molecules for cell formation and multiplication. However, Monquero
et al. (2012) reported that initial increases in the soil microbial
population from the metabolization of herbicide molecules are usually followed
by a decrease.
The results obtained in this experiment demonstrated that the
application of the commercial dose of the herbicides imazapic
(200 g a.i. ha−1) and indaziflam (100 g a.i. ha−1)
do not harm the bacterium N. amazonense
present in the inoculant. The use of herbicide molecules and formulations not
harmful to diazotrophic bacteria associated with sugarcane allows an increase
in agricultural productivity without compromising the system’s sustainability.
Results about sensitivity of sugarcane pre-sprouted seedlings inoculated
with N. amazonense to herbicide application indicated
distinct effects of PSS inoculation observed in the first assessments regarding
the impact of herbicides may be related to the complex dynamics involved in the
association between bacterium and plant and the impact of the presence of
herbicides on these organisms.
Crop-associated diazotrophic bacteria can promote biological nitrogen
fixation, siderophore synthesis, phosphate and potassium solubilization, and
the production of growth-promoting phytohormones. These microorganisms
facilitate nutrient absorption and, consequently, provide higher vigor to the
plant physiological system (Simões et al.
2018). The availability and balance in the absorption of essential nutrients
allow an increase in the plant’s capacity to carry out its metabolic functions
without undergoing damage when subjected to biotic and abiotic stresses, such
as those caused by herbicide applications (Andrade 2020).
On the other hand, the increase in absorption efficiency of the root
system can favor the absorption of herbicides present in the soil solution,
increasing the risk of phytotoxicity due to increased exposure to these
molecules (Perez 2017).
The explanation for the higher tolerance to PROTOX-inhibiting herbicides
is related to the ability of plants to metabolize peroxidative stress,
potentially through antioxidant systems (Carbonari et al. 2012). Thus,
the higher recovery of inoculated seedlings with the application of sulfentrazone may be associated with a higher physiological
plant efficiency, resulting from the beneficial action of the diazotrophic
bacterium N. amazonense.
The growth-promoting effect may be related not only to an increase in
root length but also to morphological changes in the root system. The higher
development of lateral roots and root hairs, although thinner and shorter,
allows for higher efficiency in water and nutrient absorption (Matoso et al. 2016). The biological response
regarding the inoculation of diazotrophic bacteria can be variable, as it is
related to several factors, such as plant genotype and environmental
characteristics, and there may be changes in the initial development and
allocation of biomass between different varieties and within the same variety,
considering the genotype-environment interaction (Silva et al. 2009;
Santos et al. 2012).
Imazapic stood
out negatively regarding the effects of herbicides on root length and dry
biomass, with a reduction of up to 10-times in root length relative to the
control. Imazapic acts by inhibiting the acetolactate
synthetase (ALS) enzyme, preventing the synthesis of essential amino acids
(leucine, isoleucine, and valine) and the production of new cells. The stoppage
in shoot growth and reduction in the number and length of roots are
characteristic symptoms of herbicides that have this mechanism of action (Marchi et al. 2008), which explains their
suppressive effect on the seedling roots.
The herbicide indaziflam did not interfere
with the in vitro growth of N. amazonense
at the assessed doses. Bacterial growth was inhibited by the herbicide imazapic from the recommended dose. The herbicides imazapic (200 g ai ha−1) and indaziflam (100 g ai ha−1) applied to the
soil were not harmful to N. amazonense growth.
Sugarcane pre-sprouted seedlings of the variety RB966928 were highly
susceptible to the herbicide imazapic, regardless of
the N. amazonense inoculation. Clomazone, sulfentrazone, and tebuthiuron
did not interfere with the growth-promoting effect of N. amazonense
in pre-sprouted seedlings of the variety RB966928. Inoculation with N. amazonense did not change the sensitivity of
pre-sprouted seedlings to herbicides.
Acknowledgements
The authors immensely acknowledge the financial supports from FAPESP
(Process 2020/03715-4).
Author Contributions
Luana Carolina Gomes Jonck: Formal analysis, Investigation, Methodology, writing
original draft; Marcia
Maria Rosa Magri: Conceptualization, Data curation, supervision; Patricia Andrea Monquero: Resources; Writing review and editing, Funding
Acquisition.
Conflicts of Interest
Authors declare no conflicts of interests among institutions.
Ethics Approval
Not applicable to this article.
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