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Cell colonies extracted from nodules collected from some legumes grown in 2004, 2005 and 2006 rainy seasons. The legumes include cowpea(Vigna unguiculata L. Walp), groundnuts(Arachis hypogeal L.) and soybean (Glycine max L. Merill) treated with mineral nitrogen and phosphorus fertilizers at different levels, were laid out in a randomized complete block design (RCBD) replicated three times. The isolates’ from the nodules were subjected to morphological, Gram stain and biochemical identification. Plate cultures of the isolates in a selective growth media of yeast mannitol agar (YMA) incubated at room temperature of 32oC for 72hours revealed a morphological characteristic of rhizobia cells. Morphological characteristics shows visible colonies appearing 2-4mm in diameter, circular, colourless, pulvanatate, raised elevation, smooth edge and surface, translucent viscous, musky odour. The cell morphology indicates bacterial cells were rods in chain, and dense clumps that stained gram negative by the appearance of faint pink red colour of the rod membrane walls. Inside the rods, there were small rounded granules that stained a dark purple. The biochemically identification of the cell colonies appearing smooth, white and raised colonies with a red halo streaked in a yeast mannitol agar supplemented by 2% (w/v) urea and 0.012% phenol. Rhizobia cells were also observed to appear smooth, white and raised colonies in 2days of incubation, with a red halo evidence of urea degradation confirmed rhizobia isolation. Yeast mannitol agar adjusted with dilute acid to 5.5 and with NaOH to 8.5 shows isolates suspected to be rhizobia appeared smooth, white, glistening and raised colonies of 2-4mm in diameter confirmed rhizobia presences. The isolated bacterial cell were also tested on the ability to grow in the presence of KNO3 in a yeast mannitol agar under anaerobic conditions, the appearance of raised, white colonies of 2-3mm in diameter confirmed the presences of rhizobia. The ability of rhizobial strain to tolerate NaCl were tested on tryptone yeast extract agar supplemented with 0, 1, 2 and 3% (w/v) NaCl, colonies appeared raised white, smooth and glistening 2-4mm in diameter confirmed the presences of rhizobia. Furthermore, suspected rhizobia strains tested for antibiotic resistance and a yeast mannitol agar was supplemented with 3mg/ml streptomycin the colonies appeared raised, white and glistening confirmed as rhizobia cells. |
