Isolation, Characterization and Medium Optimization of Rhizobium Symbiont(S) From Sesbania aculeata (Dhaincha)

Sesbania aculeata (Dhaincha) is a well known ‘green manure crop’ which is been widely used in organic farming as un-decomposed green manure to improve soil and crop productivity. The current study attempts to isolate and characterize (both morphologically and physiologically) Rhizobial symbionts collected from root nodules of five desi Dhaincha plants grown in five different regions of West Burdwan district, West Bengal, India. One isolate was chosen from each plant and they were designated as RW1-RW5. All of these five isolates were subjected to various biochemical tests and it was found that the strain RW3 was biochemically distinct from the other four isolates that put it in a different species category. All the five isolates were Gram negative; rod shaped bacterium and could ferment various carbohydrates as their carbon source. But it was to be noted that all the strains were weak fermenters for mannitol, the principal ingredient of the conventional YEM medium. Except RW3, all the other isolates were potent producers of amylase, an industrially important enzyme. The RW3 strain was the sole producer of cellulase, another enzyme of economic value. All the four isolates, excluding RW3 were obligate aerobic organisms giving positive results for both catalase and oxidase tests. The RW1 Rhizobial isolate was chosen at random for further physiological studies and it was found that its optimum temperature and pH for growth was 28 ^°C and & 7.0, respectively that reflected the typical features of Rhizobiaceae family. It was a non-halophile exhibiting maximum growth at only 1% concentration of NaCl. It was later subjected to large scale cultivation (5–10 liters of broth media) with a potential to be applied as a biofertilizer by designing a suitable broth medium instead of the normal YEM broth. 6% glucose yielded maximum biomass along with 1.5% MgSO₄, 2% KH₂PO₄, 1% NaCl and 6% yeast extract as indicated by A₅₄₀ of the inoculated medium after 48 hrs of growth. This modified medium such designed for biofertilizer production utilizing RW1 Rhizobial isolate could be considered for mass scale culture of the Dhaincha symbiont although field trials are recommended. Antibiotic Sensitivity Test on the RW1 strain revealed that the bacterium was most sensitive to streptomycin and kanamycin and was least affected by ampicillin as evident because of its Gram negative nature.

Five Rhizobial isolates (RW1-RW5) were isolated from five Dhaincha plants (Sesbania aculeate) form five different regions of West Burdwan district, West Bengal, India and were subjected to various biochemical tests that put the strain RW3 under a different species category owing to its distinct biochemical properties. Excluding RW3, all the four isolates were potent producers of amylase enzyme while RW3 was the only producer of cellulase enzyme. Both of these enzymes are of commercial importance.

Excluding RW3, all the other four Rhizobial strains were obligate aerobes giving positive for both catalase and oxidase tests. RW3 was only weakly positive for Oxidase teat.

All the strains were weak fermenters of mannitol, the conventional carbon source used in YEM medium.

RW1, a typical Rhizobial symbiont of the desi Dhaincha plant grows optimally at 280C temperature, pH 7.0 and at 1% concentration of NaCl. It exhibited maximum sensitivity to streptomycin and kanamycin and was the least affected by ampicillin.

A suitable production medium was optimized by modifying the conventional YEM medium for the large scale cultivation of the RW1 Rhizobial strain containing 6% glucose, 1.5% MgSO₄, 2% KH₂PO₄ and 6% Yeast Extract with 1% NaCl.