ARM Solaiman
Department of Soil Science, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur-1706, Bangladesh,
GMA Hossain
Department of Soil Science, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur-1706, Bangladesh,
MAB Mia
Department of Crop Botany, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur-1706, Bangladesh.
Isolation, Characterization, Rhizobium, Rice
Crop-Soil-Water Management
Twenty plant samples with rhizosphere soil were collected from different rice growing areas of Bangladesh to isolate Rhizobiumstrains. Samples were collected from Kushtia (AEZ-11), Rajbari (AEZ-11), Faridpur (AEZ-12) and Patuakhali (AEZ-13) districts of Bangladesh. Root with nodules from different leguminous crop viz. lentil (Lens esculentus), grass pea (Lathyrus sativus) and chickpea (Cicer arietinum) and rhizosphere soils were collected for isolation and characterization of Rhizobium strains. Samples were selected randomly from farmers’ field of the respected AEZs. Nodules separated from roots of different leguminous crops were washed in fresh water and preserved in vials containing silica gel. The collected nodules were surface sterilized. The nodules were then crushed and streaked on Yeast Extract Mannitol (YEM) agar medium contained in petridises with the help of sterile loop. Single colonies of the isolates formed on the medium were preserved for further studies A preliminary experiment was conducted in the Soil Microbiology laboratory of the Bangabandhu Sheikh Mujibur Rahman Agricultural University to characterize Rhizobium strains isolated from nodules of those crops. The strains were assessed for colony characteristics, growth rate and acid/alkali production in laboratory media with a view to know their basic properties prior to more intensive study on their performance in respect of growth and biomass production of rice. YEM containing the following constituents: K2HPO4 (0.5g), MgSO4. 7H2O (0.2g), NaCl (0.1g), CaCO3 (3.0g), FeCl3.6H2O (0.01g), Mannitol (10g), yeast extract (0.5g), agar (Difco) (10g), Congo red (02.5% solution) (10ml), deionized water to 1 litre was used. The initial pH of the medium was 7.3 which was adjusted to 7.0 by adding 0.1 N HCl solution. The medium was inoculated with the Rhizobium strains and incubated for one week. Colonies on plates were observed for their morphology and appearance. The YEM agar medium containing bromothymol blue indicator was used for identification of strains. The reaction of the rhizobial strains on this medium was noted every week up to four weeks. Fast-growing rhizobial strains produce acid in this medium, turning the medium yellow and slow growing rhizobia produce alkali which turns the medium blue 9. An in vitro experiment was conducted in the same laboratory to study the effect of Rhizobium isolates on growth and biomass production of rice. Ten of twenty isolates collected from different leguminous crops were used for the experiment. Among them four isolates viz. Le 1, Le 4, Le 6 and Le 8 were taken from lentil, four isolates viz. Ls 1, Ls 2, Ls 6 and Ls 7 were taken from grasspea and two isolates viz. Ca 3 and Ca 4 were taken from chickpea. Those isolates of rhizobia were grown in YEM broth until turbid. Rice variety BSMRAU Dhan 1 was used as the test crop. The composition of rooting solution used in this experiment was (gL- 1): KNO3 (0.505), Ca(NO3)2.4H2O (0.335), Mg SO4. 7H2O (0.37), NaNO3 (0.17), KCl (1.05×10-3), KH2PO4 (0.136), Fe-EDTA (3.55×10- 3), MnSO4.4H2O (0.81×10-3), H3BO3 (0.57×10-3), ZnSO4. 7H2O (0.22×10-3), CuSO4.5H2O (0.04×10-3), (NH4)6Mo7O24.4H2O (0.02×10-3) and distilled water- 1000 ml. Stock solution of KNO3, Ca(NO3)2.4H2O, KH2PO4, Fe-EDTA and all other micro nutrients(MnSO4.4H2O, H3BO3, ZnSO4. 7H2O, CuSO4.5H2O, (NH4)6Mo7O24.4H2O) were prepared separately10. The pH of the solution was adjusted to 6.8. Fifty five millilitre of seedling media was taken in each boiling tubes (200 mm× 20 mm). Blotting paper (50 mm long) rolls were placed at the top of the tubes and the mouth of the tubes was closed with cotton. These boiling tubes containing seedling media were autoclaved for 15 minutes at 121oC before use. The blotting paper rolls were then slid down towards the rooting solution until the top of the solution reached just below the top of the blotting paper leaving the bottom of the tubes free from blotting paper. Roots of surface sterilized, germinated seedlings (3 days old) were soaked for 30 minutes in 1 ml inoculant of specific bacteria and it was distributed among four respective test tubes (0.25 ml each of 4 test tubes). After that 1 ml of specific bacterial inoculant was added to each test tube. Inoculation of rhizobial strains were done with approximately 108 cells ml-1. With the aid of the flamed and cooled end of a forceps the surface sterilized germinated rice seedlings were transferred to each tube into a slit made on the top of the seedling media. These test-tubes were placed on packing foam by making holes on it and were covered by black paper roles from the surface of the packing foam to the top of the filter paper to exclude horizontal light from the roots. The experiment was conducted in CRD design with four replications. The rice seedlings were allowed to grow for four weeks. Data on root and shoot length, fresh and dry biomass were taken. Data on various characters of the crop were statistically analyzed to find out the significance of variation resulting from the experimental treatments. For this purpose, analysis of variance was worked out for each character of the crop. The difference between treatment means was compared by Duncan’s Multiple Range Test.
Bangladesh J Microbiol, Volume 28, Number 2, December 2011, pp 64-68
Journal