Shahnaz Sultana
Bangladesh Council of Scientific and Industrial Research (BCSIR), Dr. Qudrat-I-Khuda Road, New Elephant Rd, Dhaka, 1205, Bangladesh
Saiful Alam
Department of Microbiology, Primeasia University, Banani, Dhaka, 1213, Bangladesh
Muhammad Manjurul Karim
Department of Microbiology, University of Dhaka, Dhaka, 1000, Banglades
Agriculture, Bangladesh, PGPR, Salinity, Siderophore
Risk Management in Agriculture
Soil salinity, Plant
Bacterial isolates: Four most potential salt-tolerant candidates based on their in vitroplant growth-promoting abilities (viz.N2fixation, phosphate solubilization, and Indole acetic acid production), were screened out from soil samples of saline areas (pH>7, EC>7 dS/m), which were identified as Bacillus aryabhattaiMS3, Ochrobactrum intermediumKMS3, Ochrobactrum intermedium DMS3, and Achromobacter denitrificans. MS3 as described in our earlier study. Bacterial isolates: Four most potential salt-tolerant candidates based on their in vitroplant growth-promoting abilities (viz.N2fixation, phosphate solubilization, and Indole acetic acid production), were screened out from soil samples of saline areas (pH>7, EC>7 dS/m), which were identified as Bacillus aryabhattaiMS3, Ochrobactrum intermedium KMS3, OchrobactrumintermediumDMS3, andAchromobacter denitrificans. MS3 as described in our earlier study. Estimation of siderophore production: Siderophore production by the bacterial strains was addressed using universal chrome azurol S (CAS) and hexadecyltrimethylammonium bromide (HDTMA) agar plate that enabled qualitative detection. Briefly, the organisms were grown in minimal media, devoid of iron, but supplemented with 0.2% glucose and incubated on an orbital shaker(NEW BRUNSWICK™94 EXCELLA®E25/E25R, Germany) at 30C,120 rpm under aerobic condition. In iron-deficient conditions, the siderophore-producing bacteria would release siderophores in search of iron uptake from the medium [25]. Following 48 h of incubation, cells were pelleted by centrifugation at 13,000xg for 2 min. With the help of a cork borer, a CAS agar plate was punched to create a hole and then the collected supernatant (10μL) from each culture was taken onto the hole. After 8 h, an orange halo produced surrounding the punched surface area indicated the excretion of siderophore in the taken supernatant. The size of the discolored halo zones was recorded for each isolate. Molecular expression analysis: To observe the expression of one of the genes in the gene cluster encoding siderophore namely, entD under salt-stressed condition, the bacterial strain, B. aryabhattaiMS3 was selected and grown in two sets of 5 ml minimal media, supplemented with or without 200 mMNaCl. Each set was composed of three tubes: supplemented with (1) 0, (2)10μM, and (3) 10 mM FeCl3to observe the effect of available iron-on siderophore production. As the optimum siderophores yielding time was reported at 24 h of culture growth, the cultures were incubated on a shaker at 120 rpm for 24 h at 30ºC. The cellular RNA was extracted using RNA PurificationKit (Promega, USA) following the manufacturer's instructions. The extracted RNA was reverse transcribed into cDNA by using ImProm-II™Reverse Transcription System (PROMEGA, USA) as per the manufacture's protocol in polymerase chain reactions, done in a thermal cycler (ESCO, Singapore) under conditions of initial denaturation at 94ºC for 5 min, followed by 35 cycles of denaturation at 94ºC for 1 min 30 s, annealing at 59ºC for 1 min, and extension at 72ºC for 1 min 30 s. A final extension of10 min at 72ºC was conducted. TheentDgene was amplified using the primer pair 50- TTGGTAAAAGATGTAACGAATTGTG-30and 50-CGCCCTAAATTGTTTACTACGG-30. The amplified products were electrophoresed on 1% agarose gel and the images were captured using gel Doc (AlphaImager, USA). The images of the bands were then analyzed by ImageJ software (https://imagej.nih.gov/ij/). Statistical analysis: All the measurements were carried out multiple times. The data were built using Data Analysis Tool Pak of Microsoft Office Excel 2010. Differences were considered significant at the level of p<0.05 to compare the potential of siderophore producers in non-saline and saline conditions.
Journal of Agriculture and Food Research 4 (2021) 100150
Journal