Determination of cell size and morphological characterization of the bacterium Azotobacter sp. In the morphological characterization, transparent colonies were obtained, with an entire border, round shape, aqueous texture and convex elevation, translucent appearance and with mucus (See Fig. 1 and Table 3). According to the description of León et al. (2017) Azotobacter colonies are transparent and translucent on Winogradsky culture medium, coinciding with the results obtained in the present investigation, showing that the colonies on Winogradsky medium had a diameter of less than 1mm.
The size of the bacteria ranged between 1.35 and 1.81 µm, with an average value of cell size of 1.49 µm and its morphology is Gram negative bacillary (See Fig. 1). This size is very similar to that reported by Kennedy et al. (2005) who maintain that “the bacterial cells of Azotobacter sp. They can be rod-shaped with blunt edges to ellipsoidal and coccoid and can measure from 1.6 to 2.7 µm in diameter and from 3.0 to 7.0 µm in length”. The bacteria Azotobacter sp. are Gram negative, non-symbiotic, aerobic and diazotrophic. They grow optimally in nitrogen-free media, with phosphate, magnesium, calcium, molybdenum, iron and carbon (Heidari et al., 2018). The culture medium Winogradsky, inoculated with the bacteria, was kept in a shaking water bath (28°C/48 h).
Table 3
Characteristics of the bacterial colonies after 48 h of growth.
culture medium
|
Characteristics of the colonies after 48 h of growth
|
Diameter
|
Appearance
|
Shape
|
Edge
|
Texture
|
Elevation
|
Winogradsky
(flask )
|
< 1mm
< 0.5mm
|
translucent
|
round
|
Whole
|
watery
|
convex
|
Winogradsky
( plate )
|
1mm
|
translucent
|
round
|
Whole
|
watery
|
convex
|
† Samples stored refrigerated at 4°C in flask and plate. Source: Own elaboration. |
Bacterial concentration was performed after 48 h of growth and registered 1.8 x 10 9 cells/mL, which is similar to the number 6 according to the Mac Farland scale (See Table 4). This result coincides with Zúñiga (2012), who maintains that inoculants under laboratory conditions require an approximate concentration of 10 8 to 10 9 cells/mL for successful inoculation in plants.
Table 4
Mac Farland scale used for bacterial quantification.
No.
|
BaCl2 0.048M
V(mL)
|
H2SO4 0.36M
V(mL)
|
Vf
(mL)
|
Number of cells
|
0.5
|
0.05
|
9.95
|
10
|
0.15x10 9
|
1
|
0.1
|
9.90
|
10
|
0.30x10 9
|
2
|
0.2
|
9.80
|
10
|
0.60x10 9
|
3
|
0.3
|
9.70
|
10
|
0.90x10 9
|
4
|
0.4
|
9.60
|
10
|
1.20x10 9
|
5
|
0.5
|
9.50
|
10
|
1.50x10 9
|
6
|
0.6
|
9.40
|
10
|
1.80x10 9
|
7
|
0.7
|
9:30
|
10
|
2.10x10 9
|
8
|
0.8
|
9.20
|
10
|
2.40x10 9
|
9
|
0.9
|
9.10
|
10
|
2.70x10 9
|
10
|
1.0
|
9.00
|
10
|
3.00x10 9
|
Germination and its evaluation.
A germination test was carried out to verify the viability of the seeds. In the germination process, 100 cowpea bean seeds were used, after disinfection with a solution 4% sodium hypochlorite for 5 min, then they were washed with cold water and submerged in 500 mL of water for 1 h. In a tray, it was placed with double moistened paper towel, the seeds separated from each other by 1 cm were placed for their germination. They were sprayed with distilled water every 6 hours for 2 days so that the seeds remain moist, and 99% germination was obtained, with an average root length of 10.34 mm. On the third day, they were planted in pots for growth and subsequent evaluation.
Soil Evaluation.
The soil used for planting contained organic matter, 0.97%; electrical conductivity, 0.51dS/m; phosphorus, 7.5 ppm; potassium, 143 ppm and CEC, 16.00. It was a loam soil with 21% clay, 30% silt and 49% sand. The average soil pH was 7.50. These results are reported in Tables 5 and 7. The soil from the farm was placed in pots with a capacity of 1 kg, previously aerated one day before sowing.
The pH (potential of hydrogen) determines the degree of adsorption of ions (H+), and is the main indicator of the nutrient availability in plants, influencing the solubility, mobility, availability and other constituents and inorganic contaminants present in the soil. In this experiment, the pH in all the treatments has been maintained at a value close to 7.5, considering it slightly basic (See Table 5).
According to the edaphoclimatic requirement, the cowpea bean is considered a rustic plant and according to Arias et al. (2007) who reported that the cowpea grows better in soils with a loamy texture and does not tolerate soils with poor drainage. Its optimal pH is in the range of 6.0 to 7.5 grows in humid tropical climates and has optimum growth at temperatures between 20°C and 35°C. As stated above, the experiment carried out in this investigation had favorable conditions for the growth of cowpea beans.
According to Table 5, it is observed that using urea produced an increase in electrical conductivity from 0.51 dS/m to an average electrical conductivity of 0.97 dS/m, which is due to the hydrolysis of urea for the presence of water during irrigation and is catalyzed by the enzyme urease and because the pH of the farmland was greater than 6.3. The hydrolysis reaction occurs according to Eq. 1.
CO (NH2 )2 + H + + 2 H2 O → NH4 + + ( HCO3 ) − (1)
The ammonium Ion (NH4+) released in the hydrolysis of urea is in dynamic equilibrium with the ammonia in the atmosphere, as shown in Eq. 2:
NH4+ (adsorbed) ↔ NH4+ + OH− (sol. soil) ↔ NH3 + H2O (sol. soil) ↔ NH3 (atmosphere) (2)
The hydrolysis of urea generates a significant increase in pH around the urea granule as it consumes protons. The increase in pH displaces the balance of ammonium and ammonia, favoring the volatilization of NH3 into the atmosphere, with significant volatilization due to the slightly alkaline (pH ≈ 7.50). Therefore, the increasing of conductivity is due to the rising salinity in soil. The main cations that give rise to salinity are: sodium, calcium, magnesium and potassium and the main anions are: sulfates, chlorides, carbonates and bicarbonates.
In the treatment with urea, the decreasing of the Cation Exchange Capacity (CEC) was obtained from 16.00 to a value of 15.20 meq/100g and this is due to the fact that the ammonium released from the hydrolysis of urea is retained, in the exchange sites and there is less availability of the cation to be volatilized (See Table 7).
Moreover, when Azotobacter sp. electrical conductivity increases from 0.51 dS/m to 0.66 dS/m, being the smallest increase compared to the use of urea (See Table 6). It is observed that it passes from a slightly saline medium to a saline medium and this is due to the increasing of soluble salts in soil. The CEC increase from 16.00 to a value of 17.88 meq/100g was obtained.
The bacterium Azotobacter sp. is a nitrogen-fixing PGPR-type bacterium, which allows the transformation of N2 into bioavailable nitrogen by means of the enzyme called nitrogenase (Annan et al. 2012). This type of bacteria has the ability to solubilize phosphates (Rodriguez et al. 2006), which is why there is an increase in phosphorus from 7.5 ppm to an average phosphorus content of 8.7 ppm (See Table 5). The bacterium Azotobacter sp. has the ability to solubilize phosphates from inorganic or organic compounds, using enzymes such as non-specific phosphatases, phytases, phosphonatases and CP lyases (Lugtenberg and Kamilova 2009).
Table 5
Analysis of pH, EC, CaCO3, OM, P, K, textural class and nitrogen in the soil at the end of its evaluation of each treatment after 1 month of growth of cowpea bean.
Sample
|
pH (1:1)
|
EC (1:1)
dS/m
|
CaCO3
(%)
|
OM
%
|
P
ppm
|
K
ppm
|
Sand (%)
|
Slime (%)
|
Clay
(%)
|
Textural class
|
N
%
|
U1
|
7.51
|
0.97
|
0.10
|
1.22
|
8.1
|
146
|
49
|
28
|
23
|
Frank
|
0.05
|
U2
|
7.49
|
0.98
|
0.10
|
1.20
|
8.0
|
145
|
49
|
28
|
23
|
Frank
|
0.05
|
U3
|
7.50
|
0.96
|
0.10
|
1.22
|
8.2
|
146
|
49
|
28
|
23
|
Frank
|
0.05
|
Azo1
|
7.46
|
0.65
|
0.10
|
1.22
|
8.8
|
143
|
47
|
30
|
23
|
Frank
|
0.05
|
Azo2
|
7.55
|
0.60
|
0.10
|
1.00
|
8.7
|
143
|
47
|
30
|
23
|
Frank
|
0.04
|
Azo3
|
7.46
|
0.66
|
0.20
|
1.02
|
8.6
|
143
|
47
|
30
|
23
|
Frank
|
0.05
|
TC
|
7.50
|
0.51
|
0.10
|
0.97
|
7.5
|
143
|
49
|
30
|
21
|
Frank
|
0.04
|
Source: Own elaboration. |
Table 6
Classification of salinity based on electrical conductivity (Andrades and Martinez 2014).
EC
(dS/m)
|
EC
1/5 (dS/m)
|
Classification
|
two
|
˂0.35
|
not saline
|
2–4
|
0.35–0.65
|
slightly saline
|
4–8
|
0.65–1.15
|
Saline
|
˃ 8
|
˃1.15
|
very saline
|
Source: Own elaboration. |
Table 7
Analysis of the CEC, concentration of cations and anions in the soil at the beginning (TC) and at the end of its evaluation of each treatment after a month of growth of cowpea bean.
Sample
|
CEC
meq/100g
|
Exchangeable Cations
|
sum of cations
|
addition of bases
|
Ca+ 2
|
Mg+ 2
|
K+ 1
|
Na+ 1
|
Al+ 3 + H +
|
|
meq/100g
|
|
|
U1
|
15.04
|
11.72
|
2.50
|
0.36
|
0.46
|
0.00
|
15.04
|
15.04
|
U2
|
15.12
|
11.88
|
2.45
|
0.35
|
0.44
|
0.00
|
15.12
|
15.12
|
U3
|
15.21
|
11.92
|
2.47
|
0.34
|
0.48
|
0.00
|
15.21
|
15.21
|
Azo1
|
17.88
|
14.65
|
2.33
|
0.35
|
0.55
|
0.00
|
17.88
|
17.88
|
Azo2
|
17.12
|
14.07
|
2.22
|
0.37
|
0.46
|
0.00
|
17.12
|
17.12
|
Azo3
|
17.28
|
14.16
|
2.30
|
0.34
|
0.48
|
0.00
|
17.28
|
17.28
|
TC
|
16.00
|
13.22
|
2.10
|
0.31
|
0.37
|
0.00
|
15.04
|
15.04
|
Source: Own elaboration. |
Final evaluation of the plants
The final evaluation of bean seedlings was carried out at the end of a month of plant growth. To eliminate humidity and find the final dry weight of the plants, an oven was used at 70°C for 48 h. Tables 8 and 9 show the results of the dependent variables such as the length of the aerial part (LAP), root length (RL), fresh weight of the aerial part (FWAP), dry weight of the aerial part (DWAP), fresh weight of the root (RFW), dry weight of the root (RDW) and thickness of the stem (ST) at different dosage volumes and type of fertilizer. Likewise, the control test was carried out without fertilizer.
Table 8
Results of the response variables RFW, RDW, FWAP, DWAP and humidity at 30 days of evaluation.
Sample
|
RFW
(g)
|
RDW
(g)
|
Root
Humidity
(%)
|
FWAP
(g)
|
DWAP
(g)
|
Aerial part
Humidity
(%)
|
U1
|
0.580 ± 0.040
|
|
83.27
|
2.616 ± 0.427
|
|
86.81
|
U2
|
0.692 ± 0.084
|
0.094 ± 0.024
|
86.42
|
2.815 ± 0.623
|
|
86.18
|
U3
|
0.699 ± 0.025
|
0.097 ± 0.023
|
86.12
|
2.964 ± 0.440
|
|
86.30
|
Azo1
|
0.507 ± 0.093
|
0.082 ± 0.011
|
83.82
|
2.965 ± 0.241
|
0.369 ± 0.043
|
87.55
|
Azo2
|
0.887 ± 0.186
|
0.141 ± 0.048
|
84.10
|
3.126 ± 0.179
|
0.368 ± 0.019
|
88.23
|
Azo3
|
0.537 ± 0.168
|
0.082 ± 0.024
|
84.73
|
2.808 ± 0.726
|
0.316 ± 0.099
|
88.75
|
TC
|
0.569 ± 0.069
|
0.093 ± 0.023
|
83.66
|
2.860 ± 0.552
|
0.313 ± 0.068
|
89.06
|
Source: Own elaboration |
Table 9
Results of the LAP, RL and ST response variables at 30 days of growth.
Sample
|
LAP
(cm)
|
RL
(cm)
|
ST
(mm)
|
U1
|
14.267 ± 0.850
|
13.367 ± 0.551
|
|
U2
|
15.767 ± 0.987
|
14.567 ± 0.907
|
1.340 ± 0.381
|
U3
|
15,567 ± 1,041
|
15.100 ± 0.854
|
1.305 ± 0.350
|
Azo1
|
16.333 ± 0.757
|
11,567 ± 1,582
|
1.917 ± 0.491
|
Azo2
|
15.720 ± 0.611
|
9,520 ± 1,636
|
1.690 ± 0.477
|
Azo3
|
14,420 ± 1,342
|
9,080 ± 1,515
|
1.860 ± 0.483
|
TC
|
12,467 ± 1,102
|
8,033 ± 1,909
|
2.017 ± 0.333
|
Source: Own elaboration |
|
Figure 2 shows the growth parameters such as the length of the aerial part (LAP), root length (RL), root fresh weight (RFW), root dry weight (RDW), fresh weight of the aerial part (FWAP), dry weight of the aerial part (DWAP) and stem thickness (ST) of cowpea bean, using a fertilizer dosage of 1, 2 and 3 mL in the treatments and were compared with the control (without treatment).