Growth-promoting effects of PGPB by a single inoculation
First, the growth-promoting effects of PGPB, OFT2, and OFT5, were examined without rhizobial inoculation (leaf area, shoot dry weight, and root dry weight; Fig. 1A, 2A, C). Under 0mM NaCl condition, no negative effects of PGPB were observed in all traits, whereas a growth promotion effect was detected only in leaf area with single inoculant OFT2 as compared with non-inoculated plants (Fig. 1A). A similar trend was observed under 60 mM NaCl conditions. Significant growth enhancement was only observed in leaf area with a single inoculant OFT5 compared to non-inoculated plants (Fig. 1A, 2A, C). These results indicate that the growth-promoting ability of OFT2 and OFT5 for soybean is not necessarily effective under either 0 mM or 60 mM NaCl conditions.
Growth-promoting effects of PGPB by combined inoculation with rhizobia
First, the growth-promoting effects of rhizobia, USDA110, and SG09, were examined without inoculation with PGPB. No significant differences in leaf area, shoot dry weight, and root dry weight were observed between USDA110 and SG09 single-inoculated plants (Fig. 1B, 2B, D). PGPB inoculation increased leaf area when combined with USDA110 or SG09 under normal conditions. Consequently, USDA110 + OFT5 resulted in a significant increase in leaf area compared with USDA110. Furthermore, SG09 + OFT2 and SG09 + OFT5 significantly increased leaf area, compared to SG09 (Fig. 1B). As for shoot growth, a significant increase in shoot dry weight was observed in USDA110 + OFT5 compared to USDA110 alone (Fig. 2B). Similarly, a significant increase in shoot dry weight was observed for SG09 + OFT2 and SG09 + OFT5, compared with SG09. The root dry weight of plants inoculated with either USDA110 or SG09 was significantly higher than that of their respective rhizobial single-inoculated plants (Fig. 2D). SG09 + OFT2 and SG09 + OFT5 had beneficial effects on root dry weight, compared to the single inoculation of SG09.
Growth response to salinity stress
Compared with normal conditions, salinity stress also severely restricted all growth traits of soybeans (Fig. 1, 2). The growth-promoting effect of PGPB in response to salinity stress varied depending on the combination of rhizobia and PGPB, especially the rhizobial strain. No significant difference was observed in the growth-promoting effect of PGPB in the combined inoculation of USDA110. However, a clear and significant increase in all growth traits was detected after dual inoculation with SG09. Plants with either SG09 + OFT2 or SG09 + OFT5 showed significant or numerical growth advantages compared to SG09. In particular, plants treated with SG09 + OFT2 showed significantly improved leaf area, shoot dry weight, and root dry weight under NaCl stress conditions than plants treated with SG09 alone (Fig. 1B, Fig. 2B, D). As a general trend, the single inoculant SG09 tended to exceed the single inoculant USDA110 in all growth traits.
Salinity-induced leaf abscission
Salinity strongly induced leaf abscission in soybean regardless of rhizobial inoculation (Fig. 3, Supplementary Figure S1 online). Among the rhizobial non-inoculated groups, OFT2 significantly reduced the leaf abscission rate compared to the non-inoculated group (Fig. 3A). Dual inoculation with USDA110 + OFT2 significantly reduced the leaf abscission rate compared to the single inoculant USDA110 (Fig. 3B). However, the USDA110 + OFT5 dual inoculant did not show such an effect. The dual inoculation effect was remarkable with SG09, and 36% and 34% reductions in leaf abscission rates were observed in SG09 + OFT2 and SG09 + OFT5, respectively, compared to SG09.
Photosynthesis traits
The photosynthetic traits were significantly affected by inoculation with PGPB, rhizobial strains, and salinity stress (Table 1). The healthier plants grown under 0 mM NaCl had higher relative chlorophyll (SPAD values) and Phi2 and PhiNO ratios than more salinity-stressed plants but had a lower PhiNPQ ratio.
Table 1
Effects plant growth-promoting bacteria (PGPB) containing 1-amino-cyclopropane-1-carboxylate (ACC) deaminase on photosynthetic efficiency of photosystem II (Phi2), the yield of non-regulatory energy dissipation (PhiNO), the yield of non-photochemical quenching (PhiNPQ), and chlorophyll content (SPAD) in non-rhizobia inoculated and dual inoculated with rhizobial strains in soybean plants grown under 0 and 60 mM NaCl. Values are presented as means (S.D.), n = 4. Different letters indicate classes that show significant differences (p < 0.05) using Duncan’s Multiple Range Test (DMRT).
Traits
|
Phi2
|
PhiNO
|
PhiNPQ
|
SPAD
|
Inoculation/treatments
|
0 mM
|
60 mM
|
0 mM
|
60 mM
|
0 mM
|
60 mM
|
0 mM
|
60 mM
|
Non-Rhizobium
|
un-inoculation
|
0.31 ± 0.03ab
|
0.20 ± 0.03c
|
0.14 ± 0.05a
|
0.13 ± 0.03ab
|
0.55 ± 0.05bc
|
0.67 ± 0.05a
|
27.8 ± 2.4a
|
18.4 ± 2.4b
|
OFT2
|
0.32 ± 0.03a
|
0.25 ± 0.06bc
|
0.14 ± 0.02a
|
0.13 ± 0.04ab
|
0.55 ± 0.05bc
|
0.61 ± 0.05ab
|
28.1 ± 2.4a
|
24.4 ± 2.4a
|
OFT5
|
0.39 ± 0.04a
|
0.33 ± 0.03a
|
0.17 ± 0.02a
|
0.15 ± 0.01a
|
0.44 ± 0.05c
|
0.49 ± 0.05c
|
27.6 ± 2.4a
|
23.5 ± 2.4ab
|
Rhizobium
|
USDA110
|
0.31 ± 0.04c
|
0.11 ± 0.04e
|
0.19 ± 0.04ab
|
0.05 ± 0.02f
|
0.50 ± 0.05c
|
0.84 ± 0.05a
|
29.1 ± 2.4b
|
17.3 ± 2.4f
|
110 + OFT2
|
0.35 ± 0.03bc
|
0.23 ± 0.06d
|
0.19 ± 0.05ab
|
0.11 ± 0.01e
|
0.46 ± 0.05cd
|
0.68 ± 0.05b
|
29.4 ± 2.4b
|
21.3 ± 2.4de
|
110 + OFT5
|
0.36 ± 0.01bc
|
0.24 ± 0.08d
|
0.18 ± 0.02abc
|
0.11 ± 0.04de
|
0.46 ± 0.05cd
|
0.65 ± 0.05b
|
29.5 ± 2.4b
|
19.1 ± 2.4ef
|
SG09
|
0.35 ± 0.02bc
|
0.37 ± 0.06bc
|
0.16 ± 0.04abcd
|
0.13 ± 0.01cde
|
0.49 ± 0.05cd
|
0.50 ± 0.05c
|
29.3 ± 2.4b
|
23.8 ± 2.4cd
|
SG09 + OFT2
|
0.40 ± 0.03ab
|
0.34 ± 0.03bc
|
0.21 ± 0.06a
|
0.14 ± 0.04bcde
|
0.39 ± 0.05d
|
0.53 ± 0.05c
|
32.9 ± 2.4a
|
25.1 ± 2.4c
|
SG09 + OFT5
|
0.41 ± 0.04ab
|
0.44 ± 0.04a
|
0.19 ± 0.03ab
|
0.16 ± 0.02abcd
|
0.40 ± 0.05d
|
0.40 ± 0.05d
|
31.9 ± 2.4ab
|
24.8 ± 2.4c
|
The impact of the combined inoculation of PGPB and rhizobia on physiological traits was unclear under normal conditions. Under 60 mM NaCl conditions, a positive effect of PGPB was detected in both the USDA110 and SG09 inoculated groups. Dual inoculants USDA110 + OFT2 and USDA110 + OFT5 showed significantly higher Phi2 and lower PhiNPQ than USDA110. Similarly, SG09 + OFT5 had a higher Phi2 and lower PhiNPQ than SG09 alone. SG09 single-inoculated plants showed a relatively higher Phi2 than USDA110 single-inoculated plants under 0 mM NaCl conditions. Under 60 mM NaCl conditions, Phi2, PhiNO, and PhiNPQ significantly differed between SG09- and USDA110-inoculated plants.
Salinity also significantly decreased the SPAD values in plants. The increase in SPAD values due to OFT2 and OFT5 single inoculation tended to be inhibited by co-inoculation with USDA110. On the other hand, SG09 showed high SPAD values when inoculated alone and maintained this trend when co-inoculated with OFT2 and OFT5.
Nodulation and N2-fixation of soybean
The dual inoculant USDA110 + OFT2 resulted in a significantly greater nodule number by 29% compared with the single inoculant USDA110 (Fig. 4A). Furthermore, a significant increase in nodule number was achieved by 35% SG09 + OFT2 or SG09 + OFT5 compared with SG09. Similarly, nodule dry weight was significantly increased by dual inoculation with USDA110 + OFT5 by 24% compared to USDA110 (Fig. 4B). A similar trend was observed in the SG09-inoculation group: a significant increase in nodule dry weight by 36% with SG09 + OFT2 and 34% with SG09 + OFT5, compared to SG09. There was no significant difference in nodule number between USDA110 and SG09 (Fig. 4A). On the other hand, a significant difference in nodule dry weight between the two strains indicated that SG09-inoculated plants formed heavier nodules than plants inoculated with USDA110 (Fig. 4B). In the rhizobia-non-inoculated group, few nodules were formed on the host plants (less than 10) under normal conditions, and no nodulation was detected under 60 mM NaCl conditions (Supplementary Table S1 online).
Nodule number and dry weight were seriously limited in a saline environment. Salinity reduced nodule number by 36% in single inoculant USDA110 and 21% in single inoculant SG09, compared to their respective plants grown under normal conditions (Fig. 4A). The salinity-induced reduction in nodulation was mitigated by co-inoculation with PGPB. USDA110 + OFT2 showed significantly greater values for nodule dry weight than USDA110 alone (Fig. 4B). Among the SG09-inoculated groups, SG09 + OFT2 or SG09 + OFT5 had a considerable advantage in nodulation compared with the single inoculant SG09. A significant increase in nodule number was achieved by 35% of the plants treated with SG09 + OFT2 or SG09 + OFT5 compared with SG09 (Fig. 4A). The dual inoculation of SG09 + OFT2 benefited nodule dry weight by 39% and SG09 + OFT5 by 38% compared with SG09 alone (Fig. 4B). Salt damage affected the appearance of nodulated roots, and whitened ineffective nodules were observed (Supplementary Figure S2 D, J online). The combination of USDA110 + OFT2 suppressed the reduction of nodule number because of salinity compared to USDA110 alone (Fig. 4A). Furthermore, in the case of dual inoculation with SG09, both SG09 + OFT2 and SG09 + OFT5 recovered both nodule number and dry weight compared to SG09 (Fig. 4A, B). No significant difference in nodule number was observed between USDA110 and SG09 inoculants (Fig. 4A). Similar to the normal condition, the nodule dry weight of the single inoculant SG09 was significantly higher than that of the single inoculant USDA110 (Fig. 4B).
The dual inoculation of rhizobia and PGPB revealed a large benefit in the N2-fixation (Fig. 4C). A significant ARA enhancement of ARA was achieved by USDA110 + OFT5 relative to USDA110 under the no-salt condition. The greatest increase of more than 75% in ARA was confirmed in SG09 + OFT2 and SG09 + OFT5 plants, compared to SG09 alone. There was no significant difference in ARA between the plants inoculated with USDA110 and SG09. Under a single inoculation of rhizobia, N2- fixation in soybean plants was severely impaired by salt stress. N2-fixation efficiency in response to salinity stress depended on the rhizobia species and their combinations with PGPB. The dual inoculation of USDA110 + OFT2 gave about three times higher ARA than USDA110 alone. However, no such effect was observed for the USDA110 + OFT5. In contrast, the beneficial effects of N2-fixation under stress conditions were accompanied by dual inoculation of PGPB and SG09. SG09 + OFT2 and SG09 + OFT5 showed 112% and 74% higher ARA than SG09 alone. In the case of rhizobia, the single inoculant SG09 had a better ARA than that of USDA110.
Ethylene production
Under no stress conditions, the seedlings inoculated with OFT2 or OFT5 showed slightly lower, but no significant level of ethylene production compared to those without inoculation (Fig. 5A). NaCl treatment significantly increased ethylene production, while OFT2 and OFT5 inoculation suppressed the increase in ethylene production. Upon dual inoculation with rhizobia, the effect of PGPB was dependent on the combination of the rhizobial strains (Fig. 5B). Plants inoculated with USDA110 + OFT2 showed significantly reduced ethylene production, whereas those inoculated with USDA110 + OFT5 showed no or some reduction in ethylene levels. The effect of combined inoculation with SG09 and PGPB on reducing ethylene stress was more remarkable. Compared to single inoculation of SG09, dual inoculation of plants with SG09 + OFT2 and SG09 + OFT5 significantly reduced ethylene production.
Metal ion accumulation
The effect of PGPB on ion uptake in plants, with or without salinity, varied depending on the combination of rhizobia and PGPB (Table 2, Supplementary Table S2 online). Under normal conditions, shoot uptake (K, P, Mg, B) of SG09 + OFT2, and SG09 + OFT5 was higher than that of SG09 alone. Furthermore, the dual inoculant USDA110 + OFT5 showed higher K, Mg, Ca, and Mn uptake than the single inoculant USDA110. Under saline conditions, the lowest Na accumulation was recorded in SG09, with or without PGPB, compared to USDA110 alone.
Table 2
Effects plant growth-promoting bacteria (PGPB) containing 1-amino-cyclopropane-1-carboxylate (ACC) deaminase on shoot ions uptake in non-rhizobia inoculated and dual inoculated with rhizobial strains in soybean plants grown under 0 and 60 mM NaCl. Values are presented as means (S.D.), n = 4. Different letters indicate classes that show significant differences (p < 0.05) using Duncan’s Multiple Range Test (DMRT).
Traits
|
Na
|
K
|
P
|
Inoculation/treatments
|
0 mM
|
60 mM
|
0 mM
|
60 mM
|
0 mM
|
60 mM
|
Non-Rhizobium
|
un-inoculation
|
1.72 ± 0.79d
|
16.79 ± 3.81c
|
17.80 ± 1.41a
|
13.71 ± 1.17d
|
1.50 ± 0.14a
|
0.69 ± 0.09c
|
OFT2
|
1.65 ± 0.43d
|
26.56 ± 3.59a
|
17.11 ± 1.34ab
|
14.52 ± 1.39cd
|
1.33 ± 0.07 b
|
0.70 ± 0.08c
|
OFT5
|
1.75 ± 0.96d
|
21.02 ± 3.78 b
|
16.10 ± 0.57bc
|
15.90 ± 0.85bcd
|
1.31 ± 0.14 b
|
0.67 ± 0.11c
|
Rhizobium
|
USDA110
|
1.35 ± 0.17c
|
24.26 ± 14.25a
|
15.24 ± 1.36cde
|
11.40 ± 4.53fg
|
1.13 ± 0.04b
|
0.49 ± 0.20cd
|
110 + OFT2
|
1.61 ± 0.23c
|
12.66 ± 5.28b
|
17.95 ± 3.96abc
|
12.41 ± 2.90ef
|
1.28 ± 0.21b
|
0.48 ± 0.07cd
|
110 + OFT5
|
1.71 ± 0.55c
|
21.40 ± 3.5a
|
19.86 ± 2.53ab
|
16.61 ± 2.05g
|
1.31 ± 0.22b
|
0.37 ± 0.12d
|
SG09
|
1.69 ± 0.93c
|
13.92 ± 1.38b
|
16.87 ± 0.78bcd
|
12.41 ± 1.51ef
|
1.10 ± 0.09b
|
0.55 ± 0.09cd
|
SG09 + OFT2
|
1.28 ± 0.28c
|
12.12 ± 3.08b
|
20.44 ± 1.20ab
|
15.97 ± 1.12cde
|
1.24 ± 0.19b
|
0.66 ± 0.08c
|
SG09 + OFT5
|
1.64 ± 0.24c
|
12.60 ± 2.12b
|
20.72 ± 2.75a
|
13.72 ± 1.06def
|
1.51 ± 0.23a
|
0.58 ± 0.09cd
|
Traits
|
Mg
|
Ca
|
Mn
|
Inoculation/treatments
|
0 mM
|
60 mM
|
0 mM
|
60 mM
|
0 mM
|
60 mM
|
|
un-inoculation
|
3.09 ± 0.16a
|
2.19 ± 0.16b
|
1.08 ± 0.09a
|
0.76 ± 0.07b
|
9.42 ± 1.27a
|
6.14 ± 0.08c
|
|
OFT2
|
3.19 ± 0.25a
|
2.21 ± 0.35b
|
1.08 ± 0.09a
|
0.77 ± 0.08b
|
8.13 ± 0.76ab
|
7.15 ± 1.23bc
|
|
OFT5
|
2.97 ± 0.16a
|
2.09 ± 0.12b
|
1.04 ± 0.05a
|
0.78 ± 0.05b
|
6.75 ± 0.52bc
|
6.92 ± 0.84bc
|
|
USDA110
|
2.48 ± 0.24cd
|
1.49 ± 0.41fg
|
0.89 ± 0.10bcd
|
0.54 ± 0.05ef
|
6.67 ± 1.00cdef
|
4.80 ± 2.00ef
|
|
110 + OFT2
|
2.82 ± 0.61abc
|
1.48 ± 0.35fg
|
1.05 ± 0.20ab
|
0.58 ± 0.14ef
|
8.06 ± 1.48cd
|
5.74 ± 1.11def
|
|
110 + OFT5
|
3.21 ± 0.33a
|
1.24 ± 0.23g
|
1.17 ± 0.14a
|
0.42 ± 0.09f
|
10.93 ± 2.33a
|
4.35 ± 1.08f
|
|
SG09
|
2.61 ± 0.11bcd
|
1.60 ± 0.27fg
|
0.98 ± 0.07abc
|
0.64 ± 0.10e
|
8.46 ± 1.32bc
|
6.55 ± 1.45cdef
|
|
SG09 + OFT2
|
3.25 ± 0.23a
|
2.15 ± 012de
|
1.16 ± 0.08a
|
0.85 ± 0.04cd
|
10.77 ± 2.11ab
|
8.09 ± 0.20cd
|
|
SG09 + OFT5
|
3.25 ± 0.27ab
|
1.91 ± 0.30ef
|
1.12 ± 0.13a
|
0.71 ± 0.09de
|
8.54 ± 1.45bc
|
7.14 ± 1.36cde
|
Root colonization of PGPB and rhizobial bacteria
The colonization of Pseudomonas spp strains (OFT2 or OFT5) and Bradyrhizobium strains (USDA110 or SG09) in the roots of soybean cultivated under non-saline and saline conditions were assessed by determining the colony-forming units (CFU) per gram of root fresh weight (Fig. 6). In the non-saline condition, the population density of OFT2 and OFT5 cells in the fresh root of 14-day-old seedlings was measured at 0.97 × 104 and 1.78 × 104 CFU g− 1 fresh root, respectively. The density of soybean plants inoculated with either Bradyrhizobium strain USDA110 or SG09 exhibited a remarkable similarity. Notably, the population counts for strains USDA110 and SG09 were measured at 1.83 × 104 CFU g− 1 and 2.28 × 104 CFU g− 1, respectively.
The cell density of OFT5 did not increase when co-inoculated with either USDA110 or SG09 (Fig. 6A). However, co-inoculation of PGPB (OFT2 or OFT5) improved the colonization of Bradyrhizobium strains (USDA110 or SG09; Fig. 5B). The rhizobial cell population increased 2-3-fold in USDA110 and 2-fold in SG09, when co-inoculated with OFT5 or OFT2, respectively.
Salinity significantly decreased the colonization of Bradyrhizobium strains (USDA110 or SG09) when singly inoculated (Fig. 6B). However, no negative effect of salinity was observed for both OFT2 and OFT5 (Fig. 6A). Salinity significantly reduced the colonization of OFT5 when co-inoculated with either USDA110 or SG09 compared to non-saline conditions. On the other hand, the presence of 60 mM of NaCl significantly increased the colonization of OFT2 with either USDA110 or SG09. Salinity-induced reduction in the colonization of USDA110 was observed when co-inoculated with either OFT2 or OFT5 (Fig. 6B). However, the presence of OFT2 and OFT5 in the soybean root rescued the population of SG09 when compared with sole SG09 inoculation.
Dual inoculation efficacy between SG09 and PGPB in response to salinity
For SG09 and PGPB, which showed the highest dual inoculation efficacy, each index value was validated under salt stress conditions in an independent experiment. Multiple comparisons between inoculation conditions were performed. Similar results were obtained in the re-experiment, where dual inoculation of either SG09 + OFT2 or SG09 + OFT5 under salinity stress showed a significant improvement in the adverse effects of salinity stress on plant growth, physiological traits, nodulation, and N2-fixation (Table 3). In response to salinity stress, the reduction in growth and nodule number in the single inoculant SG09 was remarkable and was two times lower than those of SG09 + OFT2 or SG09 + OFT5.
Table 3
Effect of plant growth-promoting bacteria (PGPB) expressing 1-amino-cyclopropane-1-carboxylate (ACC) deaminase on recovery of soybean plants in terms of growth, physiological traits, and nodulation of soybean grown under salinity for 11 days conditions. Values are presented as means (S.D.), n = 4. Different letters indicate classes that show significant differences (p < 0.05) using Duncan’s Multiple Range Test (DMRT) (p < 0.05).
|
Shoot dry weight (g)
|
Root dry weight (g)
|
Chlorophyll content (SPAD)
|
Phi2
|
PhiNo
|
PhiNPQ
|
Nodule No.
|
Nodule DW (mg)
|
ARA/plant
(nL C2H4 h− 1 plant− 1)
|
SG09
|
35.96 ± 6.5a
|
2.95 ± 0.7a
|
48.00 ± 0.4a
|
0.54 ± 0.02a
|
0.23 ± 0.01a
|
0.23 ± 0.03b
|
372.25 ± 36.5a
|
816.65 ± 166.3a
|
24.76 ± 7.5a
|
SG09 + NaCl
|
7.54 ± 1.6c
|
1.16 ± 0.2b
|
29.55 ± 5.2c
|
0.22 ± 0.03c
|
0.13 ± 0.04c
|
0.59 ± 0.09a
|
152.81 ± 42.5b
|
222.50 ± 39.8c
|
12.40 ± 1.6c
|
SG09 + OFT2 + NaCl
|
15.16 ± 3.9b
|
1.73 ± 0.3b
|
38.88 ± 3.7b
|
0.3 ± 0.03b
|
0.17 ± 0.02bc
|
0.52 ± 0.04a
|
304.88 ± 38.7a
|
409.38 ± 121.1b
|
14.45 ± 2.6bc
|
SG09 + OFT5 + NaCl
|
17.28 ± 4.7b
|
1.73 ± 0.2b
|
41.93 ± 2.1b
|
0.30 ± 0.03b
|
0.18 ± 0.03ab
|
0.51 ± 0.03a
|
318.50 ± 33.2a
|
452.25 ± 84.3b
|
18.56 ± 3.0b
|