3.1. Non-phytotoxic concentrations of OEO on seeds
The impact of various OEO concentrations on soybean seeds was assessed, it was observed that at concentrations below 0.89 mg/mL, there were no effects on germination, radicle length, and chlorophyll content (Table 2). Consequently, this concentration was established as non-phytotoxic.
Table 2: Evaluated parameters of seeds treated with different concentrations of OEO.
OEO
(mg/mL)
|
% Germination
p:0,0412
|
Radicle length (cm)
p:0,0091
|
Chlorophyll content (μ g -1 fresh weight)
|
Chlorophyll a
p:0,1839
|
Chlorophyll b
p:0,2702
|
Total chlorophyll
p:0,2353
|
Control
|
90,00 bc
|
4,95 c
|
15,19 a
|
10,97 a
|
26,90 a
|
3,58
|
82,66 a
|
2,61 a
|
Nd
|
Nd
|
Nd a
|
1,76
|
96,00 c
|
3,08 b
|
Nd
|
Nd
|
Nd a
|
0,89
|
87,33 b
|
3,34 bc
|
13,37 a
|
8,33 a
|
22,18 a
|
0,45
|
90,66 bc
|
4,27 c
|
13,41 a
|
9,07 a
|
23,27 a
|
0,22
|
91,33 bc
|
4,18 c
|
13,05 a
|
8,23 a
|
21,90 a
|
Ref: nd: not determined; OEO: Oregano essential oils. Means with a p<0.05 are significantly different (distinct letters).
3.2. Effect of OEO on the germination of soybean seeds infected
On the 5th day, Germinative Energy (GE) was evaluated in seeds infected with P. syringae M7-C1 and P. savastanoi pv glycinea B076. Significant differences were noted in the initial measurement between the control seeds and those infected with both strains, which were subsequently treated with OEO. Subsequent evaluation on the 8th day, measuring Germination Power (GP), revealed notable distinctions (p: 0.0059) between the control seeds and those infected with the bacteria (B076 and M7-C1), demonstrating that the two phytopathogenic bacteria decreased the percentage of seed germination. Treatment with OEO demonstrated a slight restoration of GP in seeds infected with M7-C1. Conversely, in seeds infected with the reference strain and treated with OEO (OEO B076), no discernible differences in germination were observed compared to the control, suggesting that OEO mitigated the negative impact of the bacteria. Other parameters aligned with GP; higher GP correlated with fewer abnormal seedlings and less incidence of rotten seeds. The integrity of the seeds corresponded directly to the overall seed quality (Table 3).
Table 3: Effect of oregano EO (0.89 mg/mL) on germination parameters of seeds infected with P. syringae .
Treatments
|
5 days
|
8 days
|
Germ energy (%)
|
Germinative power (%)
|
% Abnormal seedlings
|
% Rotten seeds
|
% Hard seeds
|
Seed control
|
72,50 a
|
85,50 a
|
10,25 b
|
3,25 a
|
1,00 a
|
OEO
|
64,50 ab
|
80,00 ab
|
15,00 ab
|
4,00 a
|
1,00 a
|
B076
|
66,75 ab
|
70,00 b
|
25,50 a
|
3,25 a
|
1,25 a
|
M7-C1
|
62,50 ab
|
68,25 b
|
21,75 ab
|
7,75 a
|
2,25 a
|
OEO B076
|
53,75 b
|
73,50 ab
|
22,25 a
|
3,25 a
|
1,00 a
|
OEO M7-C1
|
60,75 ab
|
69,75 b
|
23,00 a
|
5,50 a
|
1,75 a
|
p-value (Tukey's test)
|
0,0540
|
0,0059
|
0,0066
|
0,4282
|
0,2161
|
Ref: OEO: oregano essential oil; M7-C1: P. syringae M7-C1, B076: P. savastanoi pv. glycinea B076. Means with a p<0.05 are significantly different (distinct letters).
The treatment with OEO significantly reduced the bacterial load of P. savastanoi pv. glycinea B076 in infected soybean seeds by three logarithmic units, decreasing from 1.1x10^10 CFU/mL to 1x10^7 CFU/mL in the treated samples. Similarly, OEO treatment decreased the bacterial load of P. syringae M7-C1, reducing it from 5x10^11 CFU/mL to 2.5x10^10 CFU/mL in the treated samples.
3.3. Effect of OEO on seeds infected at greenhouse scale
Growth parameters were assessed at two different intervals: 40 and 70 days after sowing, revealing no significant differences between the two-time points (p > 0.05). This indicates that there were no notable variations in plant height, seed weight, number of nodes, root length, or fresh and dry seed weight between the control group and those treated with OEO, whether infected or not. Moreover, there were no significant differences in the number and morphology of nodules between the treatments. These findings suggest that neither seed infection nor treatment with OEO exerted discernible effects on the measured growth parameters in this trial (Table 4).
Table V.4: Growth parameters (in %) of plants grown from soybeans infected with P. syringae and treated with oregano OE in greenhouse.
|
Treatments
|
Growth parameters
|
Days
|
Seed control
|
OEO
|
M7-C1
|
B076
|
OEO
M7-C1
|
OEO
B076
|
Statistical test and p-value
|
Height
|
40
|
27,60
|
27,10
|
27,60
|
24,40
|
25,00
|
25,30
|
0,8487
|
70
|
36,20
|
39,00
|
33,80
|
34,70
|
30,80
|
32,10
|
0,1454
|
Knots
|
40
|
2,00
|
1,70
|
1,90
|
1,70
|
1,90
|
1,70
|
0,3260
|
70
|
4,50
|
4,30
|
4,20
|
4,20
|
4,10
|
4,00
|
0,6216
|
Fresh weight of shoots
|
40
|
2,67
|
2,34
|
2,39
|
2,29
|
2,09
|
2,04
|
0,2008
|
70
|
5,37
|
6,26
|
4,88
|
5,45
|
5,08
|
5,13
|
0,6370
|
Dry weight of shoots
|
40
|
0,66
|
0,61
|
0,56
|
0,51
|
0,68
|
0,60
|
0,1327
|
70
|
1,57
|
2,13
|
1,40
|
1,50
|
1,38
|
1,34
|
0,0764
|
Long. Root
|
40
|
26,80
|
28,80
|
27,50
|
23,50
|
28,30
|
27,30
|
0,5640
|
70
|
31,40
|
32,00
|
29,60
|
29,50
|
29,80
|
31,40
|
0,9299
|
Fresh root weight
|
40
|
1,18
|
1,11
|
1,05
|
0,95
|
0,88
|
0,82
|
0,1532
|
70
|
1,99
|
1,87
|
1,55
|
2,53
|
2,62
|
2,19
|
0,0501
|
Dry weight of the root
|
40
|
0,37
|
0,44
|
0,38
|
0,30
|
0,28
|
0,30
|
0,0501
|
70
|
0,45
|
0,54
|
0,44
|
0,37
|
0,39
|
0,40
|
0,1842
|
Nodules
|
40
|
9,20
|
8,70
|
9,00
|
9,90
|
8,70
|
8,00
|
0,7590
|
70
|
20,00
|
18,30
|
19,20
|
17,90
|
19,10
|
18,50
|
0,08560
|
Seedling Emergence
|
10
|
86,66
|
85
|
73.3
|
75
|
76,66
|
76,66
|
-
|
Ref: OEO: oregano essential oil; M7-C1: P. syringae M7-C1, B076: P. savastanoi pv. glycinea B076. Means with a p > 0.05 are not significantly different.
When conducting the post-treatment bacteria count, a pattern akin to the germination test was observed, demonstrating a reduction in the bacterial count in seeds after OEO treatment. In P. savastanoi pv. glycinea B076, the bacterial count decreased from 1.25x10^12 CFU/mL to 1.12x10^9 CFU/mL (a reduction of 3 logarithmic units), and in P. syringae M7-C1, it decreased from 2.35x10^11 CFU/mL to 1.5x10^9 CFU/mL (a reduction of 2 logarithmic units).
At 90 days the incidence and severity parameters of bacterial blight were assessed. Figure 1 illustrates the disease incidence in soybean plants caused by P. savastanoi pv. glycinea B076, showing an incidence of 20.33%, and by P. syringae M7-C1, with an incidence of 19.67%. Treatment with OEO resulted in a reduced incidence of 17.50% in plants infected with P. savastanoi pv. glycinea B076 and 17.83% in those infected with P. syringae M7-C1. Nevertheless, no statistically significant differences were observed between the infected and treated seeds (Figure 2).
The severity of bacterial blight caused by P. syringae M7-C1 and P. savastanoi pv. glycinea B076 was recorded at 0.49% and 0.57%, respectively (Figure 2). Plants infected with P. syringae M7-C1 and treated with OEO exhibited reduced severity, with values reaching 0.36%. Similarly, in the case of P. savastanoi pv. glycinea B076 treated with OEO, a severity of 0.45% was observed. Despite the decrease in severity observed in OEO-treated plants, these differences were not found to be statistically significant when compared to the severity in infected plants.
3.4. Effect of oregano and thyme essential oils on soybean seeds infected at field scale.
Field trials were conducted to further investigate the efficacy of oregano and thyme essential oils (EO) in controlling P. syringae M7-C1. Previous research on thyme EO for greenhouse-scale trials has been documented (Sotelo et al., 2021).
At 92 days post-planting, leaf samples from plants were collected to assess the incidence and severity of the disease caused by P. syringae M7-C1 (native strain). The infection control group exhibited an incidence of 38.33% and a severity of 1.62% (Figure 5). Treatment with thyme EO (TEO) resulted in a significant reduction in both disease incidence and severity compared to the infection control group, displaying incidence values of 21.25% and severity of 0.40%. However, seeds treated with oregano EO (OEO) did not exhibit statistically significant differences from the infection control, showing an incidence of 34.07% and a severity of 1.03%.
The effect of both essential oils (EO) on seed yield was assessed by gathering data from two one-square-meter samples per treatment and replication, which involved recording the number of plants, pods, seeds, and seed weight per square meter, as well as calculating the average number of pods, seeds, and seed weight per plant for each treatment.
As illustrated in Figure 8, notable differences were observed in seed weight yield per square meter between the control group and plants infected with the phytopathogenic strain. Similar trends were observed in seeds treated with OEO, displaying a distinct deviation from the control group. However, while the seed weight was slightly lower, no statistically significant differences were evident with TEO. Interestingly, in seeds infected with the phytopathogenic strain and treated with EO, there was an increase in yield compared to untreated infected seeds, indicating a potentially positive impact of thyme EO. This increase was not significantly different from the control. These findings emphasize the influence of the bacterium on seed weight yield.
The effect of both essential oils (EO) on seed yield was assessed by gathering data from two one-square-meter samples per treatment and replication, which involved recording the number of plants, pods, seeds, and seed weight per square meter, as well as calculating the average number of pods, seeds, and seed weight per plant for each treatment.
As illustrated in Figure 8, notable differences were observed in seed weight yield per square meter between the control group and plants infected with the phytopathogenic strain. Similar trends were observed in seeds treated with OEO, displaying a distinct deviation from the control group. However, while the seed weight was slightly lower, no statistically significant differences were evident with TEO. Interestingly, in seeds infected with the phytopathogenic strain and treated with EO, there was an increase in yield compared to untreated infected seeds, indicating a potentially positive impact of thyme EO. This increase was not significantly different from the control. These findings emphasize the influence of the bacterium on seed weight yield.
Table 6: Number of plants, pods, and seeds per m2 of the field seed trial.
Treatment
|
Nº of floors
|
Nº of pods
|
Nº of seeds
|
Seed control
|
17,67 a
|
1563,17 a
|
3186,00a
|
M7-C1
|
12,67 b
|
779,17 c
|
1625,33 c
|
OEO
|
12,83 b
|
942,33 bc
|
1936,50 bc
|
TEO
|
15,83 ab
|
1201,17 b
|
2388,50 b
|
OEO M7-C1
|
13,00 b
|
1016,33 bc
|
2034,50 bc
|
TEO M7-C1
|
15,00 ab
|
1203,00 b
|
2468,33 b
|
Tukey p-value
|
0,0080
|
0,0001
|
0,0001
|
Ref: OEO: oregano essential oil; TEO: thyme essential oil, M7-C1: P. syringae M7-C1. Means with a p<0.05 are significantly different (distinct letters).
The table presented below displays the count of pods, seeds, and seed weight per plant for each treatment, aiming to evaluate variations in the emerged plants. It was evident that the bacterium not only influenced the number of plants but also detrimentally affected their yield, exhibiting notable differences particularly in the number of pods per plant (Table 7). However, the controls involving both EO and the treatments of the bacteria with these oils did not exhibit substantial differences concerning the performance of individual plants.
Table 7: Number of pods, number of seeds and weight of seeds per plant for each treatment.
Treatment
|
Nº of pods
|
Nº of seeds
|
Seed weight
|
Seed control
|
90,27 a
|
183,88 a
|
27,23 a
|
M7-C1
|
62,18 b
|
129,60 a
|
19,94 a
|
OEO
|
75.17 ab
|
155,32 a
|
24,59 a
|
OET
|
76,75 ab
|
155,98 a
|
24,41 a
|
OEO M7-C1
|
80,75 ab
|
162,77 a
|
24,77 a
|
OET M7-C1
|
80.67 ab
|
165,38 a
|
24,51 a
|
Tukey p-value
|
0,0468
|
0,1500
|
0,2625
|
Ref: OEO: oregano essential oil; TOE: thyme essential oil, M7-C1: P. syringae M7-C1. Means with a p<0.05 are significantly different (different letters).