WUEB, plant growth, and yield attributes
Microbial inoculations and water treatments significantly influenced WUEB, plant height, 1000-kernal weight, and grain yield. Root and shoot dry weight were significantly affected by microbial inoculations, water treatments, and their interactions.
WUEB, plant height (Fig. 1), root and shoot dry weight, 1000-kernal weight, and grain yield were significantly reduced with increased water stress (WW > MWS > SWS). In addition, microbial inoculations significantly enhanced WUEB, plant height, root and shoot dry weight, 1000-kernal weight, and grain yield compared to their CK.
WUEB for AMF-ACC deaminase producing PGPR co-inoculation, ACC deaminase producing PGPR, and AMF were significantly enhanced by 17.7%, 7%, and 2.3% in well water treatment (Table 1), 37.8%, 27.9%, and 20% in moderate water (Table 1), and 56%, 34.3%, and 22.1% severe water stress (Table 1) as compared to their controls. AMF-ACC deaminase producing PGPR co-inoculation, ACC deaminase producing PGPR, and AMF significantly enhanced root dry weight by 16%, 8%, and 4% in well water treatment, 21.1%, 15.8%, and 10.5% in moderate water treatment, and 42.9%, 28.26% and 21.4% in severe water stress, while upsurged grain yield by 21.1%, 5.4% and 9.7% in well water treatment, 37.3%, 20.2% and 12.3% in moderate water stress, and 63.6% 31.4% and 34.7% in severe water stress respectively as compared to control plants.
Irrespective of water treatment, AMF-ACC deaminase producing PGPR co-inoculation, ACC deaminase producing PGPR, and AMF significantly improved plant height by 12.74%, 11.03%, and 9.55%, root dry weight by 21.18%, 14.75%, and 11.04%, shoot dry weight by 16.93%, 10.88%, and 6.82%, 1000-kernal weight by 16.73%, 11.75% and 6.03% and grain yield by 19.83%, 16.15% and 10.05% as compared to non-inoculation plants.
Table 1
Effect of microbial inoculation on plant height, root and shoot dry weight, 1000-kernal weight, grain yield, and water use efficiency for biological yield (WUEB) at the harvesting stage of wheat under different water regimes
Water treatments
|
Microbial inoculation
|
Plant height (cm)
|
Root dry weight (g/plant)
|
Shoot dry weight (g/plant)
|
1000-kernal weight (g)
|
Grain yield/plant
|
WUEB (g/L H2O)
|
WW
|
AMF-PRPR
|
86.40 ab
|
0.29 a
|
3.26 a
|
42.21 a
|
4.01 a
|
2.01 a
|
PGPR
|
93.50 a
|
0.27 b
|
3.19 ab
|
39.27 b
|
3.49 bc
|
1.82 b
|
AMF
|
88.33 ab
|
0.26 bc
|
3.11 b
|
37.00 c
|
3.63 b
|
1.75 b
|
No inoculation
|
82.57 b
|
0.25 c
|
3.09 b
|
35.06 c
|
3.31 c
|
1.71 b
|
MWS
|
AMF-PRPR
|
75.53 a
|
0.23 a
|
2.89 a
|
35.99 a
|
3.13 a
|
1.96 a
|
PGPR
|
72.83 ab
|
0.22 b
|
2.61 b
|
35.45 a
|
2.74 b
|
1.82 ab
|
AMF
|
71.67 ab
|
0.21 b
|
2.52 b
|
33.17 b
|
2.56 b
|
1.70 b
|
No inoculation
|
65.33 b
|
0.19 c
|
2.24 c
|
31.58 c
|
2.28 c
|
1.42 c
|
SWS
|
AMF-PRPR
|
60.17 a
|
0.20 a
|
2.08 a
|
33.30 a
|
1.98 a
|
1.80 a
|
PGPR
|
51.50 bc
|
0.18 b
|
1.88 b
|
30.48 b
|
1.59 b
|
1.55 b
|
AMF
|
54.27 ab
|
0.17 c
|
1.71 c
|
28.64 c
|
1.63 b
|
1.41 b
|
No inoculation
|
45.90 c
|
0.14 d
|
1.51 d
|
26.21 d
|
1.21 c
|
1.16 c
|
ANOVA
|
WT
|
***
|
***
|
***
|
***
|
***
|
***
|
MI
|
***
|
***
|
***
|
***
|
***
|
***
|
WT × MI
|
ns
|
**
|
**
|
ns
|
ns
|
ns
|
Mean (n = 3) with the same letter in a column within the same water regime are statistically similar at p < 0.05 according to Duncan's multiple range test. ns indicates non-significant while **, *** indicate significance levels at 0.01 and 0.001, respectively. AMF = arbuscular mycorrhizal fungus, PGPR = Plant growth-promoting rhizobacteria, WT = Water treatments, MI = Microbial inoculation, WW = Well watered (80% FWC), MWS = Moderate water stress (50% FWC), SWS = Severe water stress (35% FWC) |
ACC accumulation
A significant microbial inoculation, water treatment, developmental stages, microbial inoculation with water treatment and developmental stages; the interaction between water treatment and developmental stages; and interaction among microbial inoculation, water treatment, and developmental stages effect observed for ACC accumulation (p < 0.001). ACC accumulation significantly increased during anthesis while significantly reduced during the pre-harvesting stage (Fig. 2a). ACC accumulation was significantly enhanced with an increase in drought stress. Microbial inoculation did not significantly influence ACC accumulation during tillering and anthesis for well-watered treatments; however, microbial treatments (AMF and PGPR co-inoculation as well as alone) significantly reduced the ACC accumulation during tillering, anthesis, and pre-harvesting stages compared to control for moderate and severe water stresses.
Mycorrhizal colonization
Mycorrhizal colonization was significantly affected by microbial inoculation, water treatment, developmental stages, microbial inoculation with water treatment and developmental stages; the interaction between water treatment and developmental stages; and interaction among microbial inoculation, water treatment, and developmental stages. Regardless of water treatments, AMF-ACC deaminase producing PGPR co-inoculation, ACC deaminase producing PGPR, and AMF averagely increased mycorrhizal colonization by 98.8%, 29.2%, and 66.5% at tillering (Fig. 2b), 112.61%, 29.06%, and 71.09% at anthesis (Fig. 2b), and 107.1%, 27.3%, and 62.2% at pre-harvesting (Fig. 2b) respectively, comparing with CK. Irrespective of developmental stages, mycorrhizal colonization in AMF-ACC deaminase producing PGPR co-inoculation, ACC deaminase producing PGPR, and AMF was 102.87%, 67.65%, and 25.49% higher in well-watered 101.46%, 56.85%, and 31.57% higher in moderate water stress, 114.33%, 73.66%, and 27.71% higher in severe water stress than that of their respective controls. Average mycorrhizal colonization was also significantly higher in severe water stress and moderate water stress compared to well-watered treatment (SWS > MWS > WW) across three growing stages.
Microbial biomass carbon and microbial biomass nitrogen
Microbial inoculation, water treatment, developmental stages, the interaction between microbial inoculation and developmental stages significantly influenced MBC (p < 0.001), while the interaction between microbial inoculation and developmental stages, water treatment and developmental stages, and water treatment and microbial inoculations also significantly influenced MBN (p < 0.001). MBC and MBN significantly upsurged during anthesis and reduced during pre-harvesting stages. MBC significantly increased with an increase in water stress (WW > MWS > SWS) (Fig. 2c), while MBN decreased in moderate water stress and enhanced in severe water stress (Fig. 2d). Microbial inoculations significantly increased MBC and MBN during three water treatments across all growth stages.
Chlorophylls and carotenoid contents
Microbial inoculation, water treatment, developmental stages, and interaction between water treatment and developmental stages significantly affected chlorophyll a content. Usually, AMF-ACC deaminase producing PGPR co-inoculation, ACC deaminase producing PGPR, and AMF had significantly higher chlorophyll a content than their respective CK in severe water stress during tillering and anthesis, while the trend was changed during pre-harvesting where chlorophyll a content was higher in well-watered treatment. In general, chlorophyll a content was increased by 9.23%, 5.87%, and 4.53% by AMF-ACC deaminase producing PGPR co-inoculation, ACC deaminase producing PGPR, and AMF, respectively, compared to their CK. Chlorophyll a content in AMF-ACC deaminase producing PGPR co-inoculation, ACC deaminase producing PGPR, and AMF, on average, was increased by 10.6%, 7.6%, and 6.7% at tillering (Fig. 3a), 8.33%, 4.95%, and 3.16% at anthesis (Fig. 3a), and 8.9%, 5.2% and 4% at pre-harvesting (Fig. 3a) in comparison with CK, respectively.
Chlorophyll b content was significantly affected by microbial inoculation, water treatment and developmental stages, and interaction between water treatment and developmental stages. Across all three developing stages, AMF-ACC deaminase producing PGPR co-inoculation, ACC deaminase producing PGPR, and AMF had significantly higher chlorophyll b content than their respective CK in severe water stress. Averagely, chlorophyll b content in AMF-ACC deaminase producing PGPR co-inoculation, ACC deaminase producing PGPR, and AMF was 26.68%, 14.66%, and 8.53% higher in well water treatment (Fig. 3b), 38.59%, 23.31%, and 15.38% higher in moderate water stress (Fig. 3b) and 56.98%, 39.19%, and 23.03% higher in severe water stress (Fig. 3b), as compared to their relative CK across three developing stages. Chlorophyll b content was increased in AMF-ACC deaminase producing PGPR co-inoculation, ACC deaminase producing PGPR, and AMF 36.4%, 23.6%, and 14.3% at tillering, 35.31%, 19.69%, and 11.57% at anthesis and 40%, 26%, and 16.5% at pre-harvesting, as compared to their corresponding CK.
Similarly, total chlorophyll was significantly affected by microbial inoculation, water treatment and developmental stages, and interaction between water treatment and developmental stages. Generally, total chlorophyll increased during anthesis, while it again reduced during the pre-harvesting stage. Total chlorophyll continued to lessen with increased drought stress across tillering and anthesis, while it again upsurges in severe stress during pre-harvesting. Total chlorophyll in AMF-ACC deaminase producing PGPR co-inoculation, ACC deaminase producing PGPR, and AMF were enhanced by 13.25%, 7.87%, and 4.90% in well water treatment (Fig. 3c), 12.81%, 8.12%, and 5.51% in moderate water stress (Fig. 3c) and 15.56%, 10.18%, and 8.12% in severe water stress (Fig. 3c), as compared to their CK respectively. During developmental stages, AMF-ACC deaminase producing PGPR co-inoculation, ACC deaminase producing PGPR, and AMF was 14.9%, 10.3%, and 7.9% higher at tillering, 13.09%, 7.55%, and 4.65% higher at anthesis and 13.5%, 8.3%, and 5.8% higher at pre-harvesting, than their corresponding CK.
Carotenoid content concentration was also significantly affected by microbial inoculation, water treatment and developmental stages, and interaction between water treatment and developmental stages. In general, carotenoids increased during anthesis and were reduced for well-watered and moderate water stress at pre-harvesting. In case of severe water stress, carotenoids continued to increase through whole developmental stages. Carotenoid content in AMF-ACC deaminase producing PGPR co-inoculation, ACC deaminase producing PGPR, and AMF were increased by 23.33%, 15.94%, and 10.73% in well-water treatment (Fig. 3d), 26.91%, 17.74%, and 11.09% in moderate water stress (Fig. 3d), and 39.39%, 27.07%, and 16.25% in severe water stress (Fig. 3d), as compared to their corresponding CK. Carotenoid content in AMF-ACC deaminase producing PGPR co-inoculation, ACC deaminase producing PGPR, and AMF was 28.1%, 18.3%, and 11.9% higher at tillering, 23.96%, 17.02%, and 10.41% increased at anthesis, and 36.7%, 24.7%, and 15.6% increased at pre-harvesting than their comparative CK.
Photosynthesis and gas exchange attributes
Microbial inoculation, water treatments, developmental stages, and interaction significantly (p < 0.05) influenced the photosynthetic rate. The photosynthetic rate increased from tillering to anthesis and decreased during pre-harvesting for well water and moderate water treatments, while there was a significant upsurge observed for severe water stress during the pre-harvesting stage. As compared to no inoculation, the photosynthetic rate of AMF-ACC deaminase producing PGPR co-inoculation, ACC deaminase producing PGPR, and AMF treatments were improved by 30.77%, 20.39%, and 14.41% in well water treatment (Fig. 4a), 36.63%, 25.97%, and 16.04% in moderate water stress (Fig. 4a) and 48.85%, 29.82% and 14.84% in severe water stress (Fig. 4a). When averaging the water treatments, AMF-ACC deaminase producing PGPR co-inoculation, ACC deaminase producing PGPR, and AMF significantly increased photosynthetic rate by 34.8%, 21.3%, and 17.6% at tillering, 43.99%, 31.27%, and 19.57% at anthesis and 35.4%, 22.4%, and 9.9% at the pre-harvesting stage, compared to that observed in non-inoculation conditions, respectively.
Microbial inoculation, water treatments, developmental stages, and their interactions significantly impacted the stomatal conductance. In general, stomatal conductance reduced with an increase in water stress (WW > MWS > SWS), while it increased with each growing stage (tillering < anthesis < pre-harvesting). Averagely, stomatal conductance was significantly upsurged for AMF-ACC deaminase producing PGPR co-inoculation, ACC deaminase producing PGPR, and AMF by 19.15%, 12.55%, and 8.19% during well water treatment (Fig. 4b), 24.59%, 15.81%, and 10.27% during moderate water stress (Fig. 4b), and 24.06%, 15.69% and 10.87% during severe water stress (Fig. 4b) than their respective controls. While stomatal conductance for AMF-ACC deaminase producing PGPR co-inoculation, ACC deaminase producing PGPR, and AMF was significantly Improved by 13.8%, 8.6%, and 5.6% at tillering, 22.86%, 14.87%, and 9.47% at anthesis and 27.9%, 18.4% and 12.7% at pre-harvesting, as compared to non-inoculated conditions.
The intracellular CO2 was significantly influenced by microbial inoculation, water treatments, developmental stages, and interactions. Intracellular CO2 reduced from tillering to anthesis, while upsurge during pre-harvesting for well water treatment, while it was increased from tillering to anthesis and reduced during pre-harvesting for both moderate and severe water stress. The intracellular CO2 for AMF-ACC deaminase producing PGPR co-inoculation, ACC deaminase producing PGPR, and AMF significantly reduced 10.5%, 7.1%, and 4.3% at tillering (Fig. 4c), 15.69%, 11.06%, and 7.14% at anthesis (Fig. 4c) and 15.4%, 11.2%, and 7.3% during pre-harvesting (Fig. 4c), relative to their respective controls.
Similarly, Microbial inoculation, water treatments, developmental stages, and their interactions significantly influenced the transpiration rate. The general trend was similar to stomatal conductance, where it lessened with an escalation in water stress (WW > MWS > SWS), while it amplified with each growing stage (tillering < anthesis < pre-harvesting). The transpiration rate, averagely, for AMF-ACC deaminase producing PGPR co-inoculation, ACC deaminase producing PGPR, and AMF was increased by 20.75%, 13.74%, and 9.73% during well water treatment (Fig. 4d), 21.26%, 14.55%, and 9.23% during moderate water stress (Fig. 4d), and 46.41%, 35.84%, and 27.5% during severe water stress (Fig. 4d) as compared to their relative non-inoculations.
Principal component analysis (PCA)
Dim 1 explained 60.4%, 59.1%, and 45.9%, and dim 2 explained 28.2%, 23.0%, and 31.1% of the total variance in well-watered, moderate water stress, and severe water stress. Figure 5 showed that intracellular carbon dioxide was significantly away from photosynthesis, transpiration, and stomatal conductance with increased water stress. ACC accumulation was significantly away from root colonization, transpiration, and stomatal conductance during well-watered treatment (Fig. 5a), root colonization, chlorophyll contents, transpiration, stomatal conductance, and photosynthesis during moderate water stress (Fig. 5b), and significantly away from root colonization, microbial biomass carbon and nitrogen, photosynthesis, transpiration, stomatal conductance, chlorophyll content, and carotenoids during severe water stress (Fig. 5c).