2.5. Characterization of plant growth promoting traits
Siderophore productions of isolates were determined by the Chrome Azurole S (CAS) method of Schwyn and Neilands (1987). The production of siderophore was indicated by a change in color of the medium from blue to orange. Phosphate solubilizing activity of isolates was evaluated on Sperber medium: 0.5 g L−1 yeast extract, 0.1 g L−1 CaCl2, 0.25 g L−1 MgSO4·7H2O, 2.5 g L−1 Ca3(PO4)2, 10 g L−1 glucose, 15 g L−1 agar (Sperber, 1958). The medium was spot inoculated with 7 μL of inocula and incubated at 28 °C for 7 days. The development of a clear zone around the bacteria was taken as an index of phosphate solubilization. It was computed as the ratio of total diameter (colony + halo zone) to colony diameter (Edi-Premono et al., 1996). Isolates were inoculated in nutrient broth containing 2.5 g L−1 Ca3(PO4)2 and incubated at 27 °C in shaker incubator for seven days. Each day the change in pH in the broth culture was determined with a pH meter.
Isolates were also screened for the productions of hydrogen cyanide (HCN) using NGA-plate amended with 4.4 g L−1 glycine following the method described in Lorck (1948). A Whatman filter paper No.1 soaked in 2% sodium carbonate in 0.5% picric acid solution was placed at the top of the plate. Plates were sealed with Parafilm and incubated at 28 °C for four days. Development of orange to red color indicated HCN production.
The productions of indole acetic acid (IAA) by the isolates were determined following the method of Bric et al. (1991). A 48 h old test bacterial culture grown on NGA-plate was inoculated into nutrient broth supplied with 3 mM tryptophan and incubated at 28 °C for 48 h. Bacterial cells were centrifuged at 3000 rpm for 30 min. 2 mL of the supernatant were mixed with 100 μL of ortho-phosphoric acid and 4 mL of Solawaski’s reagent (50 mL 35% perchloric acid; 1 mL 0.5 M FeCl3) and incubated for 30 min. Development of a pink color indicates IAA production. The pink color was quantified using a spectrophotometer (Beckmann DU640, USA) at 535 nm. The concentrations of IAA produced by isolates were determined using a standard curve prepared from pure IAA.
2.5. Characterization of plant growth promoting traitsSiderophore productions of isolates were determined by the Chrome Azurole S (CAS) method of Schwyn and Neilands (1987). The production of siderophore was indicated by a change in color of the medium from blue to orange. Phosphate solubilizing activity of isolates was evaluated on Sperber medium: 0.5 g L−1 yeast extract, 0.1 g L−1 CaCl2, 0.25 g L−1 MgSO4·7H2O, 2.5 g L−1 Ca3(PO4)2, 10 g L−1 glucose, 15 g L−1 agar (Sperber, 1958). The medium was spot inoculated with 7 μL of inocula and incubated at 28 °C for 7 days. The development of a clear zone around the bacteria was taken as an index of phosphate solubilization. It was computed as the ratio of total diameter (colony + halo zone) to colony diameter (Edi-Premono et al., 1996). Isolates were inoculated in nutrient broth containing 2.5 g L−1 Ca3(PO4)2 and incubated at 27 °C in shaker incubator for seven days. Each day the change in pH in the broth culture was determined with a pH meter.Isolates were also screened for the productions of hydrogen cyanide (HCN) using NGA-plate amended with 4.4 g L−1 glycine following the method described in Lorck (1948). A Whatman filter paper No.1 soaked in 2% sodium carbonate in 0.5% picric acid solution was placed at the top of the plate. Plates were sealed with Parafilm and incubated at 28 °C for four days. Development of orange to red color indicated HCN production.The productions of indole acetic acid (IAA) by the isolates were determined following the method of Bric et al. (1991). A 48 h old test bacterial culture grown on NGA-plate was inoculated into nutrient broth supplied with 3 mM tryptophan and incubated at 28 °C for 48 h. Bacterial cells were centrifuged at 3000 rpm for 30 min. 2 mL of the supernatant were mixed with 100 μL of ortho-phosphoric acid and 4 mL of Solawaski’s reagent (50 mL 35% perchloric acid; 1 mL 0.5 M FeCl3) and incubated for 30 min. Development of a pink color indicates IAA production. The pink color was quantified using a spectrophotometer (Beckmann DU640, USA) at 535 nm. The concentrations of IAA produced by isolates were determined using a standard curve prepared from pure IAA.
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2.5. Characterization of plant growth promoting traits
Siderophore productions of isolates were determined by the Chrome Azurole S (CAS) method of Schwyn and Neilands (1987). The production of siderophore was indicated by a change in color of the medium from blue to orange. Phosphate solubilizing activity of isolates was evaluated on Sperber medium: 0.5 g L−1 yeast extract, 0.1 g L−1 CaCl2, 0.25 g L−1 MgSO4·7H2O, 2.5 g L−1 Ca3(PO4)2, 10 g L−1 glucose, 15 g L−1 agar (Sperber, 1958). The medium was spot inoculated with 7 μL of inocula and incubated at 28 °C for 7 days. The development of a clear zone around the bacteria was taken as an index of phosphate solubilization. It was computed as the ratio of total diameter (colony + halo zone) to colony diameter (Edi-Premono et al., 1996). Isolates were inoculated in nutrient broth containing 2.5 g L−1 Ca3(PO4)2 and incubated at 27 °C in shaker incubator for seven days. Each day the change in pH in the broth culture was determined with a pH meter.
Isolates were also screened for the productions of hydrogen cyanide (HCN) using NGA-plate amended with 4.4 g L−1 glycine following the method described in Lorck (1948). A Whatman filter paper No.1 soaked in 2% sodium carbonate in 0.5% picric acid solution was placed at the top of the plate. Plates were sealed with Parafilm and incubated at 28 °C for four days. Development of orange to red color indicated HCN production.
The productions of indole acetic acid (IAA) by the isolates were determined following the method of Bric et al. (1991). A 48 h old test bacterial culture grown on NGA-plate was inoculated into nutrient broth supplied with 3 mM tryptophan and incubated at 28 °C for 48 h. Bacterial cells were centrifuged at 3000 rpm for 30 min. 2 mL of the supernatant were mixed with 100 μL of ortho-phosphoric acid and 4 mL of Solawaski’s reagent (50 mL 35% perchloric acid; 1 mL 0.5 M FeCl3) and incubated for 30 min. Development of a pink color indicates IAA production. The pink color was quantified using a spectrophotometer (Beckmann DU640, USA) at 535 nm. The concentrations of IAA produced by isolates were determined using a standard curve prepared from pure IAA.
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