Isolation , Characterization and Selection of Rhizobium Strains for Cultivation of Sesbania sp

The experiment was conducted to isolate Rhizobium form the root nodules of Deshi dhaincha (Sesbania aculeate) and African dhaincha (Sesbania rostrata), characterization of isolates for authentication, testing their performance as the inoculants and selection of better performed strains for future use as biofertilizer. Nineteen rhizobial strains were isolated from root nodule of dhaincha from 5 Agro Ecological Zones (AEZ 9, AEZ 11, AEZ 12, AEZ 13 and AEZ 28) of Bangladesh. The strains were characterized through morphological, biochemical, microscopic and growth observation. Experiment was conducted in bacteriologically controlled condition using sterile sand in plastic pot in glasshouse. Two varieties of Sesbania named Deshi dhaincha and African dhaincha were used as test crop varieties. Five plants were grown in each plastic pot. After 25 days of sowing plants were harvested and data on nitrogen content of shoot, shoot dry weight, root dry weight, nodule number and nodule dry weight of Sesbania cultivars in glass house were collected. Rhizobial inoculation had significant and positive effect on nitrogen content of shoot, shoot dry weight, root dry weight, nodule number and nodule dry weight for both varieties of Sesbania in sterile condition. The strain DhM3 recorded the highest nitrogen content of shoot, shoot dry weight, root dry weight, nodule number and nodule dry weight. The strain DhSk2 ranked second and DhSk4 ranked third in respect of growth, nodulation, nitrogen fixation and biomass production. Therefore, these three strains can be used for Rhizobium inocula production for cultivation of Sesbania but field level extended studies recommended


I. Introduction
Soils with adequate fertility status are essential for crop productivity and sustainability.Leguminous plants proved to be useful tool for improving soil fertility through regenerative means.Leguminous plants in association with Rhizobium can fix significant amount of atmospheric nitrogen from air which contributes to soil nitrogen pool (Jefing et al., 1992).On a global level, annual contribution of biological nitrogen fixation has been estimated about 172 million tons.Legumes contribute about 25% (33 m tons) of biologically fixed nitrogen, which is slightly less than that supplied to agroecosystems through chemical fertilizers (Azam, 2001;Lshizuka, 1992).Sesbania being potential in nitrogen fixing bacteria is cultivated in Bangladesh.Its inoculation with superior rhizobial strains is essentially required to increase the yield of legumes through nitrogen fixation (Athar, 1998).

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Published with open access at http://www.journalbinet.com/Rhizobium-legume symbiosis can increase yield with subsequent decrease in pollution (Freiberg et al., 1997).Rhizobial isolates vary in their nitrogen fixation potential and in improving the vegetative and reproductive growth of different crops under varying environmental conditions (Shishidu and Pepper, 1990;Gopalakrishnan and Grish, 1999).So, effective nitrogen fixing strains of rhizobia are being developed as inoculant for various legumes (Hardarson, 1993;Brockwell and Bottomely, 1995;Shah et al., 2000).This research was conducted to achieve two major objectivesfirstly, isolation and characterization of rhizobia from Deshi and African dhaincha and secondly, the effect of isolated rhizobia inoculants on growth, nodulation and biomass production of Deshi dhaincha (Sesbania aculeata) and African dhaincha (Sesbania rostrata) plants.
Larger, healthy and mature nodules were selected.After separation nodules were washed in running tap water and transferred to sterile vial.Selected nodules were then immersed into 70% alcohol solution for 30 seconds and in 3% hydrogen peroxide solution for 3 minutes.Hydrogen peroxide treated nodules were then rinsed with sterile mild hot water for several times.Nodules were then cut transversely with sterile blade and the milky suspension directly from the cut nodule was streaked out on congo red yeast extract mannitol agar (CRYEMA) plates using inoculation needle under laminar flow hood.Plates were incubated for 7 days at 28º C temperature.
Colonies were characterized through a systematic series of tests such as cultural tests (i.e.shape, size, opacity, elevation, surface, margin, consistency, emulsifiability, colour odour etc.), microscopic tests (i.e.simple staining, gram staining, motility observation etc.), biochemical tests (i.e.Congo red absorption test, BTB test, Hofer's alkaline broth test etc.) and nodulation tests.
Nodulation capacity of rhizobial isolates were determined by growing plants in plastic buckets containing 2 kg autoclaved sand (at 20 lbs pressure for 2 hours) in glass houses.Seeds of both Deshi dhaincha (Sesbania aculeata) and African dhaincha (Sesbania rostrata), were rinsed with concentrated Sulphuric Acid (H2SO4) for few seconds and immediately washed thoroughly by distilled water.Prior to sowing, seeds were treated with a thick Rhizobium suspension of each isolate.Ten seeds were planted in each bucket and thinned to keep five plants after germination.Sterilized Jensen's (1942) seedling solution was added to each bucket at one day intervals.
Plants samples were collected from the plastic buckets at 25 days of sowing.From each buckets 3 randomly selected plants were carefully taken out so that no nodule was left in the soil.Roots were washed and the Nodules from the root system of each plant were separately collected and counted.Oven dry weights of nodules were also recorded.
Data obtained from the pot experiment were analyzed statistically by F-test to examine whether treatment effects were significant or insignificant (Gomez and Gomez, 1984).The treatment means were evaluated by Duncan's New Multiple Range Test (DMRT).

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All the isolates were found short rod in shape, motile in nature and gram negative in reaction.The bacterial isolates did not absorb congo red at young stage but absorbed slightly when cultures became old.All the bacterial isolates produced acid in variable amounts on BTB-YEMA plates.Among the nineteen isolates none had grown on Hofer's alkaline broth.
All the isolates showed good growth at temperature 28 0 C and 32 0 C. Most of the isolates grew weakly at 14 0 C. At 22 0 C most isolates exhibited medium growth while seven isolates (DhT1, DhT2, DhSk2, DhSk3, DhP1, DhP2 and DhP3) recorded weak growth.All the isolates grew at 38 0 C.Only nine isolates (DhF1, DhF2, DhT1, DhT2, DhSk1, DhSk2, DhSk3, DhSk4 and DhP1) showed good growth at 38 0 C while rest ten showed medium growth.Most of the isolates do not grew at 45 0 C.

Table 01 . Effect of Rhizobium inoculation on nitrogen content of shoot, shoot dry weight, root dry weight, nodule number and nodule dry weight of Sesbania sp
In a column, figures having similar letter (s) do not differ significantly as per Duncan's Multiple Range Test (DMRT).
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Table 02 . Interaction effect of Rhizobium inoculation on nitrogen content of shoot, shoot dry weight, root dry weight, nodule number and nodule dry weight of Sesbania sp
In a column, figures having similar letter (s) do not differ significantly as per DMRT.