Introduction

Biological nitrogen fixation is an important aspect of nutrition in crop production throughout the world .The process saves a huge sum of money which otherwise would have to be pumped  agricultural productivity to consistently sustain an increasing world population. Among various symbiotic association of biological nitrogen fixation, the Rhizobium legume association is the most common in both temperate and tropical climates.  Rhizobia  plays a great role in nature as they are greatest N₂ fixers, particularly in leguminous plants and leguminous plants are the predominant source of supplying protein to the population of developing countries like India. (1,2).

Looking to the need of location specific strain of Rhizobia to increase yield of pulses and other leguminous plants the investigations were concentrated on isolation and identifying these local strains for effective nodulation. (3). The performance of these locally isolated strains of Rhizobia depends up on soil environment, competitor microbes and other rhizospheric factors. In the present study most efficient local strain of Rhizobia (UR5) from Marathwada region of Maharashtra state was assessed for its nodulation capacity per plant, Urd bean is cultivated on a large scale now a days in this region and carrier based inoculants are widely used in the recent times. However, various factors on efficacy of this strain are already studied (4). This paper describes the performance of the locally isolated strain of Rhizobium with different carriers on Urd bean nodulation and increase in dry weight.  

Materials and Methods

Isolation and maintenance of Rhizobium

The Urd bean seeds cv.AU4 were collected from local market. The strain RhizobiumUR5 isolated from Chudawa, Parbhani Dist .of Maharashtra state (5) of Marathwada.  Rhizobia were isolated and maintained on YEMA medium either by sub-culturing at frequent intervals or as lyophilized cultured kept at 5 °C this system maximizes the storage period and minimizes genetic variations and contamination. The culture was also dried on porcelain beads over a desiccant in screw cap bottles. The authenticity check was carried out regularly. Their effectiveness on nodulation capacity and dry matter of Urd bean was tested. In earlier studies, strain Rhizobium UR5 from Chudawa village of Dist. Parbhani, Maharashtra, India was found very much effective .Their capacity to nodulate along with some carrier such as lime pelleting, Jaggery, lignite, sawdust, sterilized cow dung powder was tested by usual methods(3). The number of nodules and dry matter of plant along with different inoculants were evaluated in a pot culture experiment for three years i.e. 2008-2011.

Screening and culturing of Rhizobium

Cryema Test: 5 ml. of Congo red dye was mixed with a one litre of YEMA medium to prepare CRYEMA medium. Bacterial colonies on the YEMA medium were streaked on the CRYEMA medium and the petriplates were incubated at 28+ 2⁰ C for 5-7 days. Rhizobial cells forms white circular, entire, raised, convex colonies, The white colonies were picked up to produce Rhizobium inoculants.

Microscopic Observation: Bacterial cells in the CRYEMA medium were stained with carbol fuschin and visualized under a compound microscope. This dye stains the B-polyhdroxybutyrate granule in the The cells of those colonies having B-polyhdroxybutyrate granule were picked up to establish Rhizobium inoculants.

Glucose Peptone Agar Test (GPA Test): Rhizobial colonies were streaked on YEMA medium and a master plate was made. colonies in the master plates were transferred to GPA medium by replica plating .Those colonies in the master plates fail to grow GPA medium belong to rhizobia .This test was confirmative test to test the purity of Rhizobial (4)

Inoculation: The surface sterilized seeds were used for inoculation. The seeds were dried in shade and sown in earthen pots of respective treatments. These pots were watered with an interval of two days or on when required. After 15 days of sowing the thinning was done and five plants were maintained in each pot.

The observations were recorded for number of nodules and dry matter. Plants were uprooted carefully washed and number of nodules per plant were recorded, The nitrogen content was determined by Microkjaldhal’s method .To identify the best method of inoculation, a pot culture experiment was conducted using different method of inoculation. The strain UR5 which was found superior was used. Medium type of soil was sterilized in autoclove at 30 lbs for two hours and used in the experiments. The Urd bean seeds were surface sterilized by treating with 0.1% mercuric chloride.

Carriers

The Urd bean seeds were inoculated using following carriers –

Lime pelleting

The seeds of Urdbean were inoculated with Rhizobium species coated with lime to form a pellets.

Rhizobium culture and jaggery

Seeds were dipped in the slurry of jaggery and Rhizobia broth and dried in shade and used for sowing.

Rhizobium culture alone

Broth culture of Rhizobium was diluted with sterile distilled water and the seeds were soaked in it. The seeds were air dried in shade and used for sowing.

Rhizobium culture and jaggery with saw dust/cow dung/lignite

The Rhizobium broth culture was grown for seven days. The cow /cow dung /lignite/saw dust was grinded to fine powder of 0.01mm size. The powder was mixed with the Rhizobium broth culture separately .After thorough mixing the powder was dried in shade under aseptic conditions and was used separately for inoculation.In control treatment the seeds were soaked in sterile water, dried in shade and were sown in pots. The observations were recorded on nodule number and dry weight of whole plant on 40^th day of sowing. The plants were grown for 40 days in uniform condition along with different inoculants. After 40 days the number of nodules per plants was recorded and the dry matter of plant was estimated by usual method. The experiment was done in three sets each and for three consecutive years i.e. 2008-2011,

Data were subjected to Analysis of Variance(ANOVA) depending upon experimental design following the procedure as given by Gomez and Gomez(6).

Result and Discussion

The effect of the Rhizobium UR5 inoculation on nodulation of Urd bean by using various carrier is presented in table 1. The result indicated that the local Rhizobium strain UR5 performed well when jaggery was used as carrier and the number of nodules was significantly higher than other carriers used. The trend in results recorded during 2008-2009, 2009-2010, 2010-2011 was similar.

Table 1: Effect Of Carrier By Strain Rhizobium Ur5 On Nodulation Of Urd Bean.

The effect of the Rhizobium UR5 inoculation on dry weight of Urd bean by using various carrier is presented in table 2. The result showed that the local Rhizobium strain UR5 performed well when jaggery was used as carrier. The dry weight recorded was significantly higher than other carriers used. The trend in results recorded during three consecutive years was similar.

Table 2:  Effect Of Carrier By Strain Rhizobium Ur5 On Dry Weight Of Urd Bean Plants.

 Thus the results showed that this local strain seems to be very effective in Urd bean when it is used for inoculation along with jaggery. The process of infection of plants by Rhizobia is very much competitive (7). The jaggery slurry method was more successful for inoculation, because in some plants the invasion of bacteria requires synthesis of important exo-polysaccharides (Marketoni.et.al., 2002) .Further investigation on local strains of bacteria and determining biodiversity of microbes is essential so that Rhizobia can be effectively utilized as Bio-fertilizers.

References 

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2. Randhawa, G.S., Shubha, Singh, N.,K., Kumar, A, Bhalla, A. (2003). Recent advances in Rhizobium-legume symbiosis. Indian J Exp Biol. 41(10):1184-97.
3. Rajbhoj B.G. and O.S.Rathor. (2011) Diversity of Rhizobium and effects on nodulation of Urd bean. Bioinfolet. 08: 93-95.
4. Rajbhoj B.G. and O.S.Rathor.(2010) Bioprospecting potential of some local Rhizobia from Marathwada, Mahrashtra state. The Biosphere. 2(2):161-162.
5. Rajbhoj B.G. and O.S.Rathor. (2011) Diversity of Rhizobium and effects on nodulation of Urd bean. Bioinfolet. 08: 93-95.
6. Gomez K.A.et al[1984].Statistical procedures for agricultural research.2^nd Ed. Wiley, New York.680 pp.
7. Campbell, G.R., Sharypova, L.A., Scheidle H, Jones, K.M., Niehaus, K., Becker, A., Walker, G.C. (2003) Striking complexity of lipopolysaccharide defects in a collection of Sinorhizobium meliloti mutants. J. Bacteriol. 185(13):3853-62.
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