Istiaq Ahmed
Department of Agricultural Chemistry, Faculty of Agriculture, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh
Md. Tofazzal Islam
Department of Biotechnology, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Salna, Gazipur, Bangladesh
Md. Akhter Hossain Chowdhury
Department of Agricultural Chemistry, Faculty of Agriculture, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh
Md. Kamruzzaman
Plant Breeding Division, Bangladesh Institute of Nuclear Agriculture (BINA), Mymensingh-2202. Bangladesh
Arsenic, Bacteria, Germination and growth, Rice
Agricultural Chemistry Laboratory, Faculty of Agriculture, BAU, Mymensingh-2202, Bangladesh
Risk Management in Agriculture
Performance
Soil sample collection and bacteria isolation: Arsenic contaminated soils were collected from the two locations of Faridpur near Dumrakandi and Chandpur near Nagda Bazar, Matlab under AEZ 12 and 17, respectively, of Bangladesh. Surface soil (0 to 15 cm depth) were collected, placed in plastic bag and kept at 4ºC until further analysis. Some physio-chemical parameters of soil like pH, arsenic content (μg g-1), available P (μg g-1), available S (μg g-1), exchangeable K (meq/100 g soil) were measured. The BSMY I medium was used for the isolation of bacteria from As contaminated soil samples contained the following ingredients (g L-1): 1g yeast extract, 0.3g (NH4)2SO4, 0.14g MgSO4. 7H2O, 0.2g CaCl2.2H2O, 0.1g NaCl, 0.05g KH2PO4, 0.05g K2HPO4, 0.0006g H3BO3, 0.00017g CoCl2.6H2O, 0.00009g CuCl2.2H2O, 0.0001g MnCl2.4H2O, 0.00022g ZnCl2, 10g glucose. The ingredients were dissolved in Tris-HCl buffer and pH of the medium was adjusted at 8.0 before autoclaving the medium. The BSMY II medium was prepared to study growth phase of bacteria using the following ingredients (g L-1): 1g yeast extract, 0.25 g NH4Cl, 0.62 g MgCl2.6H2O, 0.15 g CaCl2.2H2O, 0.1 g NaCl, 0.14 g KH2PO4, 0.5 g KCl, 0.00006 g H3BO3, 0.00012 g CoCl2.6H2O, 0.000015 g CuCl2.2H2O, 0.0001 g MnCl2.4H2O, 0.00022 g ZnCl2, 10 g glucose. The ingredients were dissolved in deionized water adjusted to pH 6.8 with NaHCO3. Two grams of each soil sample was dissolved in 20 mL 0.9% NaCl and shaken for 3 minutes. Then 5 mL of soil suspension was inoculated into 50 mL BSMY I containing 5 mM sodium-arsenite and incubated at 25ºC on rotary shaker at 120 rpm for 3 days for isolation of As resistant bacteria. Five mL of culture was transferred into fresh BSMY I medium containing 10 mM sodiumarsenite, and transferred twice into new medium that was supplemented with 20 and 40 mM sodium-arsenite. After growth was observed, 0.1 ml of culture was spreaded on BSMY II agar that contained 40 mM sodiumarsenite and incubated at 25ºC for 3 days. Bacterial isolates that could tolerate the highest As concentration were selected and identified by their morphological features and biochemical properties. Individual colonies of isolated strains of BTLs were picked from BSYM II agar medium and streaked on NBA medium for their mass growth. The culture of each isolate was stored in the 20% glycerol at -20ºC for further studies. Gram staining reaction: Bacterial cultures (after 24 hours incubation) for each isolate were taken to perform the gram reaction test. Bacterial mass were taken out from culture, spread on clean glass slide followed by drying in air without heat and was flamed it lightly and gently to settle the bacteria. Crystal violet was then poured over the bacterial smear on the slide for 2-3 minutes and washed in tap water for 1 minute and lightly blot dry on a paper towel to remove crystal violet. The smear was flooded with iodine solution for 1 minute and washed in tap water for few seconds and then blot dried. It was decolorized with solvent e.g. alcohol, until the solvent flowed colorlessly from the slide (about 2-4 seconds) and rinse in tap water for 5 seconds. Then the smear was counterstained for about 1 minute with safranin solution and washed briefly in tap water followed by blot dry. One drop emulsion oil was then added on the slide and placed it on compound microscope with 100x magnification for observation. Potassium hydroxide solubility test: A loopful of bacteria was picked from a well grown colony with a sterilized toothpick and mixed with a drop of 3% aqueous potassium hydroxide solution on a glass slide for less than l0 seconds. After mixing, the toothpick was raised a few centimeters from the glass slide. Strands of viscid material confirmed the bacterium was gram-negative. Growth of the bacterial isolates at varying pH: The arsenic resistant bacteria isolates were grown in BSMY II broth cultures with varying levels of pH (5.5 to 8.5) in a incubator shaker up to 48 hours at 25ºC to see their growth. The optical density of the broth culture was recorded by a spectrophotometer (PD-3003 UV, APEL) at 600 nm to measure the growth of the bacterial strains. Each treatment was replicated three times and data were expressed as the mean value. Effects of bacterial inoculation on germination and seedling growth of rice: The test tubes for this in-vitro culture of the rice seedlings were washed with detergents and rinsed with deionized water and then autoclaved for the sterilization. Two strains of arsenic resistant bacteria (resistant to 40 mM NaAsO2) were selected from the previously isolated arsenic resistant bacterial strains and designated by BTL0011 and BTL0022, respectively. They were cultured in BSMY I broth medium for 3 days at 25 ± 1ºC temperatures in a shaker at 120 rpm. When growth was found optimum; the cultures were checked for purity and population. Then these were centrifuged at 5500 rpm repeatedly with deionized distilled water for three times. Then these bacterial cells without any culture media were suspended in sterilized water. These bacterial suspensions were used for seed inoculation of rice for in-vitro culture. The selected varieties of rice were BRRI dhan29 and BRRI dhan47. Seeds were surface sterilized by using 70% ethanol for 10 minutes, 1% NaOCl for 1 minute and 100% ethanol for five minutes, respectively after every step washed by distilled water for five times. The seeds were then soaked in the bacterial suspension for 12 hours. After that, these seeds coated with bacteria were kept in blotting paper for absorbing extra aqueous mass from the seeds. These seeds were then used for in-vitro culture. The bacteria coated seeds were placed on agar media taken into test tubes (18 × 1.6 cm) for in-vitro culture. The conducted experiment was a three factor experiment viz. rice varieties, arsenic resistant bacterial inoculants and different concentration level of As. The agar-water containing no arsenic and no arsenic resistant bacteria was used as control. Different concentration levels of As were 5 ppm, 30 ppm, 45 ppm and 60 ppm. The plant height (shoot and root length of seedlings) were measured 7 days after seed sowing. The in vitro experiment was laid out in CRD (Completely Randomized Design) with 3 replications. Statistical analysis: The statistical analysis of the experiment was carried out using statistical computer package MSTAT-C. The ANOVA followed by Duncan’s Multiple Range Test (DMRT) were to be analyzed for the present study.
Res. Agric. Livest. Fish. Vol. 2, No. 2: 229-237; (2015)
Journal