Effects of Salt Stress on Cell Surface Properties and Symbiotic Performance of Root Nodulating Bacteria

In the current study fifty root nodulating bacterial strains isolated from Cicer arietinum (Chickpea) and Vigna radiata (Mungbean) nodules were subjected to different NaCl concentrations. Out of these 20 salt tolerant isolates were selected for the production of cell surface component like glucan, lipopolysaccharide, exopolysaccharide and motility under salt stress condition. Symbiotic performance of Vigna radiata and Cicer arietinum were done under salt stress condition by using 4 isolates (CMR3,CMR13,MR5.MR11) which showed the tolerant behavior in all cell surface properties and one sensitive isolate (MR30) was used for comparision.All growth parameters of selected legumes were enhanced at 5% salt concentration as compare to control in CMR3,CMR13,MR5.MR11. A decline in all parameters was observed in isolate that does not show good cell surface components under salt stress. This shows the inhibition of cell surface properties under salt stress, affects the symbiotic performance of both parameters negatively. The present study concludes that under salt stress conditions, tolerant strains successfully overcome the stressful environmental conditions by maintaining the factors, essential for symbiosis in an ideal system.


Introduction
Soil salinity is one of the significant environmental problem facing agricultural production in many areas and affects approximately 7% of the world's total land area 1 .Salinity decreases plant growth and yield, depending on the plant species, salinity levels, and ionic composition of the salts 2 .Legume establishment and its growth in field is greatly depends on nitrogen fixation and nodulation in legume-rhizobium symbiotic relationship.This symbiotic association are unfavourably affected by soil salinity.
Saline behavior of many cultivated crops varies in different ways and depends on environmental factors like climatic conditions, soil properties, and the stage of growth 3 Rhizobia are more economically grows on many substrates [4][5][6] Rhizobial surface components contributes important parameter for deciding the perfect relation between host and plant for bringing about the infection that leads to nodulation and nitrogen fixation. 7

2.3.1Test for beta-glucan
Isolates were streaked on yeast extract mannitol (YEM )plates having aniline blue of 1mg/ml.They were given different salt stress (5%, 5.5% up to 9.5%).After proper incubation those show blue coloured colony were considered to be beta glucan positive and white colored colony were negative.

Test for production of lipopolysaccharide
All isolates were streaked on tryptone yeast extract (TY )having sodium deoxycholate at the rate of 1mg/ml.They were given different salt stress (5%, 5.5% up to 9.5%).After proper incubation those showed growth were lipopolysaccharide (LPS) producers.

Test for the production of exopolysaccharide
All cultures were streaked on yeast extract mannitol (YEM) plates having congo red dye at the rate of 1mg/ml.They were given different salt stress(5%, 5.5% up to 9.5%)..Those shows reddish pink mucoid growth were considered to be EPS positive and white coloured were negative.

Swarming activity
Cultures were spotted on swarm plates having tryptone yeast extract (TY) medium with 0.3% agar.They were given different salt stress (5%, 5.5% up to 9.5%).The motility of bacterial strains were determined by the spread of the colonies in the swarm plates.

Symbiotic properties
Pot culture experiment was done according to Vincent (1970) 8

Statistical Analysis
Data analysis was done using SPSS version 12.0

Isolation and Biochemical characteristics of isolates from root nodule of Cicer arietinum (Chickpea) and Vigna radiata (Mungbean)
In this study symbiotic performance of nodule isolated bacteria in relation to their salt stress tolerance was investigated.Previous investigations regarding such analysis under stress conditions have also been carried out 11 .

.3.2 Test for production of Exo-polysaccharide (EPS)
Exo-polysaccharide synthesis appears to be a common feature

Test for production of lipopolysaccharide (LPS)
Salt-stress responses also lead to changes in lipopolysaccharide, 15 and is important components of the external cell wall of gram negative bacteria, may play a major role in the adaptation of bacteria to the environment.LPS 16

Swarming behavior of Mungbean and Chickpea isolates under salt stress
Swarming behavior is the marker for the motility and chemotaxis.
Chemotaxis and motility improved chemical or physical contact with the root, enhanced potentially suitable sites for infection and fast infection thread development .Our results showed that swarming activity of few strains goes on increasing up to 5% salt and thereafter with an increase in salt concentration in the medium it decreases, only five isolates obtain swarming behaviour at 9.5% salt concentration (Table 2)

Symbiotic association under different salt concentration
Since cell-surface components of Rhizobium help in nodulation and nitrogen fixation, therefore four salt tolerant strains with the ability to produce cell-surface components at all concentrations of salt (5.0%-9.5%)were selected for the study of their symbiotic performance with their host plants under salt stress.One strain selected to check the effect of cell surface molecules of Rhizobium on productivity of their host plant that could not produce a cell-surface component under salt stress condition.
Nodulation was adversely affected by salinity treatment.The nodule per plant decreases with increasing Nacl levels.The maximum number of nodule per plant was found in MR30 at control treatment and minimum number of nodule per plant was observed in CMR3 at 5% Nacl treatment (Fig 5).The higher level of salt concentration (6%-9.5%)completely inhibited nodule formation Elsheik and Wood(1995) 18 observed that growth and nodulation of soyabean were adversely affected by salinity and nodulation was more sensitive than plant growth to salinity.

3. 2
Fig 1a Effect of salt concentration on isolates of Chickpea (Cicer arietinum) Fig 2a Effect of beta-glucan on isolates on Chickpea (Cicer arietinum) Fig 4a Effect of LPS on isolates of Chickpea (Cicer arietinum)

Fig 5 Fig 6
Fig 5 Effect of salt concentration on root nodule of Chickpea (Cicer arietinum) and Mungbean (Vigna radiata) All other tested parameters of salt tolerant isolates were enhanced in soil treated with 5% Nacl concentration, then there was a significant decrease in all parameters with the increase in salt concentration from 5.5%-9.5%.While in case of salt sensitive isolate (MR30) all parameters was decreased with the increase in concentration of salt (Fig 6-Fig 13).Data analysis showed that results are significant at P=0.05% level.

and method UK Journal of Pharmaceutical and Biosciences Available at www.ukjpb.com
growing demands of pulses in agriculture two leguminous pulses i.e.Chickpea(Cicer arietinum) and Mungbean(Vigna radiata) are selected for studying the salt stress changes in Mesorhizobium and Rhizobium legume symbiosis.2 Materials ISSN: 2347-9442 Singh et al.Effects of Salt Stress on Cell Surface Properties UK J Pharm & Biosci, 2015: 3(1); 24 2.1 Isolation and characterization of root nodulating bacteria Bacterial strains were isolated from the root nodule of Cicer arietinum (Chickpea) and Vigna radiata (Mungbean) according to