Out of 23 initially screened rhizobacteria (Table 7 ) isolated from wheat rhizospheric soil, collected from Old Shujabad Road (30.11°N and 71.43°E) and Akramabad (30.16°N and 71.29°E), four most efficient drought-tolerant ACC-deaminase producing PGPR were screened out after a laboratory hydroponic trial in the Department of Soil Science, Bahauddin Zakariya University Multan, Pakistan. For screening of most effective drought tolerant PGPR strains, polyethylene glycol 6000 (PEG-6000) was used (0, 10 and 20%) to maintain osmotic potential (0.05, −0.23 and −0.78 MPa) to introduce drought stress63 (link),64 .
Molecular identification of the most efficient drought tolerant ACC deaminase producing PGPR was done by 16S rRNA gene sequencing using PCR primers 1492R 5′ (TAC GGY TAC CTT GTT ACG ACT T) 3′ and 27F 5′ (AGA GTT TGA TCM TGG CTC AG) 3′. The gene sequencing primers were 907R 5′ (CCG TCA ATT CMT TTR AGT TT) 3′ and 785F 5′ (GGA TTA GAT ACC CTG GTA) 3′. Finally, 16S rRNA gene sequences were aligned and relationships were deduced using BLAST analysis65 (link). Most efficient drought-tolerant ACC-deaminase producing PGPR were identified as AbW1 = Leclercia adecarboxylata (NR_104933.1), CbW2 = Agrobacterium fabrum (NR_074266.1), CbW3 = Bacillus amyloliquefaciens (FN_597644.1) and AbW5 = Pseudomonas aeruginosa (CP012001.1). These PGPR strains were able to grow at the osmotic potential −0.78 MPa generated through 20% polyethylene glycol 6000 (PEG). The DF minimal salt medium (4.0 g KH2PO4, 6.0 g Na2HPO4, 0.2 g, MgSO4.7H2O, 2.0 g glucose, 2.0 g gluconic acid and 2.0 g citric acid with trace elements: 1 mg FeSO4.7H2O, 10 mg H3BO3, 11.19 mg MnSO4.H2O, 124.6 mg ZnSO4.7H2O, 78.22 mg CuSO4.5H2O, 10 mg MoO3, pH = 7.2 and 0.5 M ACC as a sole nitrogen source) was used to grow the strains66 (link). For determination of indole acetic acid (IAA) with and without L-tryptophan, Glickmann and Dessaux67 (link) method was adopted.
To confirm the presence of AcdS gene that plays key role in synthesis of ACC deaminase NCBI gene bank was consulted. From NCBI gene bank it was confirmed that B. amyloliquefaciens (https://www.ncbi.nlm.nih.gov/nuccore/KX709841.1/ ), A. fabrum (https://www.ncbi.nlm.nih.gov/protein/PZP48640.1/ ) and P. aeruginosa (https://www.ncbi.nlm.nih.gov/nuccore/CP014948.1/ ) have AcdS gene while work is yet continued on L. adecarboxylata. For assessing the ACC deaminase produced by PGPR methodology of El-Tarabily68 (link) and Honma and Shimomura20 was used. Pikovskaya’s medium was used to examine the phosphorus solubilizing activity of PGPR as described by Vazquez et al.69 (link). Potassium solubilizing activity of PGPR was assessed according to the methodology of Candra Setiawati and Mutmainnah70 . Characterization of the PGPR isolates is provided in Table 7 .
For the production of biochar, timber waste was collected from local timber market. The timber waste was initially sun-dried and then pyrolyzed at 389 °C for 80 min in an especially designed pyrolyzer as described by Qayyum et al.27 . All the pyrolyzed material (biochar) was then crushed in a grinder and passed through 2 mm sieve. Finally, the fine powder of timber waste biochar (BC) was stored in air tight plastic jars27 .
The pH and ECe of BC were determined by mixing the BC and water with the ration, 1:20 (w/v) as described by Qayyum et al.27 . Di-acid (HNO3: HClO4) digestion71 of biochar was done for the analysis of total phosphorus following yellow color method on spectrophotometer72 , and those of potassium and sodium on flame photometer73 . For the determination of nitrogen, H2SO4 digestion72 was done followed by distillation on Kjeldahl’s distillation apparatus74 . The volatile matter and ash content of biochar were analyzed according to Qayyum et al.75 (link) by heating the biochar in muffle furnace at 450 °C and 550 °C respectively. The fixed carbon in biochar was assessed (Table6 ) using the equation as follows76 ; The plastic bag (30 cm deep × 20 cm in diameter) was used as a pot, having capacity to carry 8 kg soil. The soil was collected from the plough layer of bank of the Chenab River, Multan, Punjab, Pakistan. The soil of selected area was previously characterized as dark yellowish brown, moderately calcareous, weakly structured and well drained with Cambic subsurface horizon and an Ochric epipedon77 (link). The soil texture was determined by hydrometer method78 which was sandy loam (USDA triangle) with mixed hyperthermic Haplocambids. The organic matter in soil was determined by Walkley79 (link). The organic nitrogen in soil was determined using the equation: For extractable soil P determination, Olsen and Sommers80 method was used. Similarly, the extractable K in soil was determined according to the method described by Nadeem et al.73 (Table 6 ).
The seeds of wheat (Galaxy-2013) were obtained from the certified seed dealer of the Government of Punjab, Pakistan. Healthy seeds were separated from broken and weak seeds. The seeds were surface-sterilized with sodium hypochlorite (5%) followed by 3 washes with ethanol (95%). Finally, all the seeds were washed three times with sterilized deionized water82 (link). For PGPR inoculation, 10 ml of inoculum (0.5 optical density at 535 nm wavelength)83 (link) was added along 10% sugar (glucose) in 100 g sterilized seeds. After proper mixing of seeds, inoculum and sugar solution, top dressing of seeds was done with a mixture of peat and clay (3:1 ratio) as described by Ahmad et al.84 (link). Before inoculation of seeds, the peat and clay mixture was sterilized at 121 °C for 20 min in an autoclave83 (link). All the control treatment seeds were also top dressed with peat and clay mixture along with 10% sugar solution without inoculum85 (link).
In each pot, 10 seeds of wheat were initially sown. In control, the soil normal moisture (NM) was maintained at the level of 70% of field capacity (FC70) throughout the experiment on weight basis. However, to introduce mild drought (MD) and severe drought (SD) stress as per treatment plan, the soil moisture was maintained at the level of 50% and 30% of field capacity (FC50 and FC30), respectively, throughout the trial as suggested by Boutraa et al.86 (link). After germination of seeds, five healthy seedlings were kept in each pot by thinning.
The pot experiment was conducted in the research area of the Department of Soil Science, Bahauddin Zakariya University Multan, Pakistan under drought stress on wheat. There were 15 treatments with 3 replications, following factorial completely randomized design (CRD). The treatments included: Control (No PGPR + No BC), L. adecarboxylata, A. fabrum, P. aeruginosa, B. amyloliquefaciens, 1BC, L. adecarboxylata + 1BC, A. fabrum + 1BC, P. aeruginosa + 1BC, B. amyloliquefaciens + 1BC, 2BC, L. adecarboxylata + 2BC, A. fabrum + 2BC, P. aeruginosa + 2BC and B. amyloliquefaciens + 2BC.
Leaf gas exchange parameters (net photosynthetic rate, net transpiration rate and stomatal conductance) were determined with the help of Infra-Red Gas Analyzer (CI-340 Photosynthesis system, CID, Inc. USA) by joining 4 leaves of wheat. On a sunny day, the readings were taken between 10:30 and 11:30 AM at saturating intensity of light87 (link).
After 50 days of sowing, the seedlings were harvested from each pot for the measurement of shoot length and determination of electrolyte leakage, proline contents, photosynthetic pigments level and nutrients concentration in the shoot.
The electrolyte leakage (EL) was determined following the procedure given by Lutts et al.88 (link). The leaves were washed with deionized water and then cut using a steel cylinder having diameter 1 cm. One gram of uniform sized leaf pieces were immersed in a test tube containing deionized water (20 ml) and incubated at 25 °C for 24 h. The electrical conductivity (EC1) was determined using pre-calibrated EC meter. The second EC (EC2) was noted heating the test tubes in a water bath at 120 °C for 20 min. The final value of EL was calculated using the equation as follows;
For proline assessment in wheat leaves, methodology stated by Bates et al.89 (link) was followed. The proline was extracted from fresh (0.1 g) leaves in 2 ml of 40% methanol. After extraction, the 1 ml mixture of glacial acetic acid and 6 M orthophosphoric acid (3:2 v/v) was mixed in 1 ml extract along with 25 mg ninhydrin. Then the solution was incubated at 100 °C for 60 min. After cooling down, 5 ml Toluene was added. For the estimation of proline contents, absorbance was noted on spectrophotometer at 520 nm wavelength.
The chlorophyll a, chlorophyll b and total chlorophyll contents were determined in the fresh leaves of wheat according to the protocol given by Arnon90 (link). The extract was taken from the leaves using acetone (80%) solution. For the estimation of chlorophyll a and chlorophyll b, the absorbance was taken at 663 and 645 nm wavelength, respectively on spectrophotometer. Final calculations were made using the following relations; where, OD = Optical density (wavelength). V = Final volume made. W = Fresh leaf made (g).
The samples were digested with sulfuric acid72 followed by distillation on Kjeldahl’s distillation apparatus74 . The yellow colour method was used for the determination of phosphorus concentration noting absorbance at 420 nm on spectrophotometer72 . As far as the K concentration in wheat shoot and grain is concerned, the samples were digested and then run on flame photometer as described by Nadeem et al.73 .
The wheat plants were harvested after 125 days of sowing for the determination of grains yield pot−1, straw yield pot−1 and 100-grain weight. Weight of 100 grains, straw and grains yield pot−1 were assessed on top weight balance. For straw yield, plants were harvested at 4 inches above the ground surface. Sun dried 100 grains of wheat were counted randomly and manually and then weighed on top weight balance. Total wheat grains collected from a single pot were weighed and considered as grain yield pot−1.
Statistical analyses of the data were carried out using standard statistical procedures91 . All the treatments were compared using Tukey’s test at p ≤ 0.05.
Molecular identification of the most efficient drought tolerant ACC deaminase producing PGPR was done by 16S rRNA gene sequencing using PCR primers 1492R 5′ (TAC GGY TAC CTT GTT ACG ACT T) 3′ and 27F 5′ (AGA GTT TGA TCM TGG CTC AG) 3′. The gene sequencing primers were 907R 5′ (CCG TCA ATT CMT TTR AGT TT) 3′ and 785F 5′ (GGA TTA GAT ACC CTG GTA) 3′. Finally, 16S rRNA gene sequences were aligned and relationships were deduced using BLAST analysis65 (link). Most efficient drought-tolerant ACC-deaminase producing PGPR were identified as AbW1 = Leclercia adecarboxylata (NR_104933.1), CbW2 = Agrobacterium fabrum (NR_074266.1), CbW3 = Bacillus amyloliquefaciens (FN_597644.1) and AbW5 = Pseudomonas aeruginosa (CP012001.1). These PGPR strains were able to grow at the osmotic potential −0.78 MPa generated through 20% polyethylene glycol 6000 (PEG). The DF minimal salt medium (4.0 g KH2PO4, 6.0 g Na2HPO4, 0.2 g, MgSO4.7H2O, 2.0 g glucose, 2.0 g gluconic acid and 2.0 g citric acid with trace elements: 1 mg FeSO4.7H2O, 10 mg H3BO3, 11.19 mg MnSO4.H2O, 124.6 mg ZnSO4.7H2O, 78.22 mg CuSO4.5H2O, 10 mg MoO3, pH = 7.2 and 0.5 M ACC as a sole nitrogen source) was used to grow the strains66 (link). For determination of indole acetic acid (IAA) with and without L-tryptophan, Glickmann and Dessaux67 (link) method was adopted.
To confirm the presence of AcdS gene that plays key role in synthesis of ACC deaminase NCBI gene bank was consulted. From NCBI gene bank it was confirmed that B. amyloliquefaciens (
For the production of biochar, timber waste was collected from local timber market. The timber waste was initially sun-dried and then pyrolyzed at 389 °C for 80 min in an especially designed pyrolyzer as described by Qayyum et al.27 . All the pyrolyzed material (biochar) was then crushed in a grinder and passed through 2 mm sieve. Finally, the fine powder of timber waste biochar (BC) was stored in air tight plastic jars27 .
The pH and ECe of BC were determined by mixing the BC and water with the ration, 1:20 (w/v) as described by Qayyum et al.27 . Di-acid (HNO3: HClO4) digestion71 of biochar was done for the analysis of total phosphorus following yellow color method on spectrophotometer72 , and those of potassium and sodium on flame photometer73 . For the determination of nitrogen, H2SO4 digestion72 was done followed by distillation on Kjeldahl’s distillation apparatus74 . The volatile matter and ash content of biochar were analyzed according to Qayyum et al.75 (link) by heating the biochar in muffle furnace at 450 °C and 550 °C respectively. The fixed carbon in biochar was assessed (Table
Characteristics of soil, timber waste biochar (BC).
| Experiment | Biochar | Unit | Value | ||
|---|---|---|---|---|---|
| Attriburs | Unit | Value | |||
| Sand | % | 55 | pH | — | 7.03 |
| Silt | % | 30 | ECe | dS m−1 | 0.89 |
| Clay | % | 15 | Volatile Matter | % | 30.26 |
| Texture | Sandy Loam | Ash Content | % | 10.19 | |
| pHs | — | 8.43 | Fixed Carbon | % | 59.55 |
| ECe | dS m−1 | 1.95 | Total N | % | 0.29 |
| Organic Matter | % | 0.45 | Total P | % | 0.53 |
| Organic N | % | 0.023 | Total K | % | 1.36 |
| Extractable P | mg kg−1 | 8.16 | Total Na | % | 0.28 |
| Extractable K | mg kg−1 | 204 | |||
Characteristics of PGPR.
| Isolates | PGPR traits | ||||
|---|---|---|---|---|---|
| IAA | IAA | ACC deaminase | Phosphorus solubilization | Potassium solubilization | |
| BbW6 | — | — | 104.2 ± 10.9 | — | 12.4 ± 1.22 |
| BbW12 | — | — | 94.9 ± 6.91 | — | — |
| AbW4 | 0.86 ± 0.07 | 8.11 ± 1.26 | 131.3 ± 10.1 | — | 10.6 ± 1.91 |
| CbW4 | 0.66 ± 0.12 | 9.52 ± 0.60 | 84.2 ± 7.19 | 6.22 ± 0.34 | 13.7 ± 1.63 |
| AbW1 | 3.42 ± 0.27 | 67.8 ± 2.20 | 304.9 ± 24.1 | 26.6 ± 1.04 | 20.1 ± 1.02 |
| BbW9 | 0.62 ± 0.06 | 7.33 ± 0.40 | 134.6 ± 20.6 | 10.2 ± 0.22 | 14.5 ± 1.58 |
| AbW9 | — | — | 94.7 ± 15.3 | — | 9.84 ± 1.33 |
| AbW8 | 0.16 ± 0.04 | 2.14 ± 0.17 | 181.2 ± 259 | 9.41 ± 0.29 | 11.7 ± 1.26 |
| CbW3 | 1.12 ± 0.60 | 17.3 ± 2.34 | 313.2 ± 34.3 | 20.9 ± 2.48 | 23.4 ± 1.92 |
| AbW16 | — | — | 144.3 ± 23.2 | 11.2 ± 0.12 | 15.6 ± 1.20 |
| CbW5 | — | — | 153.5 ± 21.7 | 10.6 ± 0.27 | 13.3 ± 1.18 |
| CbW2 | 2.43 ± 0.34 | 58.8 ± 3.27 | 349.6 ± 21.4 | 16.2 ± 1.48 | 26.7 ± 1.49 |
| BbW14 | — | — | 149.6 ± 11.1 | 9.84 ± 0.10 | 14.7 ± 1.38 |
| AbW3 | — | — | 209.2 ± 29.4 | — | 11.9 ± 1.61 |
| BbW8 | 0.12 ± 0.04 | 3.44 ± 0.37 | 179.3 ± 26.8 | 8.21 ± 0.38 | — |
| AbW20 | — | — | 172.0 ± 20.1 | 11.4 ± 0.22 | 13.6 ± 1.73 |
| CbW6 | 0.36 ± 0.02 | 1.52 ± 0.35 | 159.6 ± 31.3 | 7.43 ± 0.19 | 15.2 ± 1.56 |
| AbW5 | 3.16 ± 0.21 | 24.8 ± 1.49 | 245.4 ± 19.5 | 22.8 ± 1.36 | 17.9 ± 1.02 |
| BbW4 | 0.56 ± 0.11 | 6.14 ± 1.06 | 349.6 ± 21.4 | — | 11.6 ± 1.44 |
| BbW10 | — | — | 119.7 ± 24.9 | 13.4 ± 0.24 | — |
| AbW11 | — | — | 194.7 ± 10.6 | 12.8 ± 0.29 | — |
| AbW2 | 0.76 ± 0.05 | 14.7 ± 1.09 | 129.6 ± 7.46 | 13.0 ± 0.35 | 10.9 ± 1.41 |
| CbW7 | 0.46 ± 0.09 | 10.4 ± 1.16 | 89.4 ± 10.1 | 11.9 ± 0.12 | 10.3 ± 1.28 |
The seeds of wheat (Galaxy-2013) were obtained from the certified seed dealer of the Government of Punjab, Pakistan. Healthy seeds were separated from broken and weak seeds. The seeds were surface-sterilized with sodium hypochlorite (5%) followed by 3 washes with ethanol (95%). Finally, all the seeds were washed three times with sterilized deionized water82 (link). For PGPR inoculation, 10 ml of inoculum (0.5 optical density at 535 nm wavelength)83 (link) was added along 10% sugar (glucose) in 100 g sterilized seeds. After proper mixing of seeds, inoculum and sugar solution, top dressing of seeds was done with a mixture of peat and clay (3:1 ratio) as described by Ahmad et al.84 (link). Before inoculation of seeds, the peat and clay mixture was sterilized at 121 °C for 20 min in an autoclave83 (link). All the control treatment seeds were also top dressed with peat and clay mixture along with 10% sugar solution without inoculum85 (link).
In each pot, 10 seeds of wheat were initially sown. In control, the soil normal moisture (NM) was maintained at the level of 70% of field capacity (FC70) throughout the experiment on weight basis. However, to introduce mild drought (MD) and severe drought (SD) stress as per treatment plan, the soil moisture was maintained at the level of 50% and 30% of field capacity (FC50 and FC30), respectively, throughout the trial as suggested by Boutraa et al.86 (link). After germination of seeds, five healthy seedlings were kept in each pot by thinning.
The pot experiment was conducted in the research area of the Department of Soil Science, Bahauddin Zakariya University Multan, Pakistan under drought stress on wheat. There were 15 treatments with 3 replications, following factorial completely randomized design (CRD). The treatments included: Control (No PGPR + No BC), L. adecarboxylata, A. fabrum, P. aeruginosa, B. amyloliquefaciens, 1BC, L. adecarboxylata + 1BC, A. fabrum + 1BC, P. aeruginosa + 1BC, B. amyloliquefaciens + 1BC, 2BC, L. adecarboxylata + 2BC, A. fabrum + 2BC, P. aeruginosa + 2BC and B. amyloliquefaciens + 2BC.
Leaf gas exchange parameters (net photosynthetic rate, net transpiration rate and stomatal conductance) were determined with the help of Infra-Red Gas Analyzer (CI-340 Photosynthesis system, CID, Inc. USA) by joining 4 leaves of wheat. On a sunny day, the readings were taken between 10:30 and 11:30 AM at saturating intensity of light87 (link).
After 50 days of sowing, the seedlings were harvested from each pot for the measurement of shoot length and determination of electrolyte leakage, proline contents, photosynthetic pigments level and nutrients concentration in the shoot.
The electrolyte leakage (EL) was determined following the procedure given by Lutts et al.88 (link). The leaves were washed with deionized water and then cut using a steel cylinder having diameter 1 cm. One gram of uniform sized leaf pieces were immersed in a test tube containing deionized water (20 ml) and incubated at 25 °C for 24 h. The electrical conductivity (EC1) was determined using pre-calibrated EC meter. The second EC (EC2) was noted heating the test tubes in a water bath at 120 °C for 20 min. The final value of EL was calculated using the equation as follows;
For proline assessment in wheat leaves, methodology stated by Bates et al.89 (link) was followed. The proline was extracted from fresh (0.1 g) leaves in 2 ml of 40% methanol. After extraction, the 1 ml mixture of glacial acetic acid and 6 M orthophosphoric acid (3:2 v/v) was mixed in 1 ml extract along with 25 mg ninhydrin. Then the solution was incubated at 100 °C for 60 min. After cooling down, 5 ml Toluene was added. For the estimation of proline contents, absorbance was noted on spectrophotometer at 520 nm wavelength.
The chlorophyll a, chlorophyll b and total chlorophyll contents were determined in the fresh leaves of wheat according to the protocol given by Arnon90 (link). The extract was taken from the leaves using acetone (80%) solution. For the estimation of chlorophyll a and chlorophyll b, the absorbance was taken at 663 and 645 nm wavelength, respectively on spectrophotometer. Final calculations were made using the following relations; where, OD = Optical density (wavelength). V = Final volume made. W = Fresh leaf made (g).
The samples were digested with sulfuric acid72 followed by distillation on Kjeldahl’s distillation apparatus74 . The yellow colour method was used for the determination of phosphorus concentration noting absorbance at 420 nm on spectrophotometer72 . As far as the K concentration in wheat shoot and grain is concerned, the samples were digested and then run on flame photometer as described by Nadeem et al.73 .
The wheat plants were harvested after 125 days of sowing for the determination of grains yield pot−1, straw yield pot−1 and 100-grain weight. Weight of 100 grains, straw and grains yield pot−1 were assessed on top weight balance. For straw yield, plants were harvested at 4 inches above the ground surface. Sun dried 100 grains of wheat were counted randomly and manually and then weighed on top weight balance. Total wheat grains collected from a single pot were weighed and considered as grain yield pot−1.
Statistical analyses of the data were carried out using standard statistical procedures91 . All the treatments were compared using Tukey’s test at p ≤ 0.05.
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