SALT TOLERANCE AND BIOCHEMICAL CHARACTERIZATION OF RHIZOBIA ISOLATED FROM SOME WILD CROTALARIA SPP

Kranti Balasaheb Patil and * Shivaji Sopanrao Kamble. Mycology and Plant pathology laboratory, Department of Botany, Shivaji University, Kolhapur. 416 004 MS (India). ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

1819 alcohol for 10 seconds and then with 0.1% HgCl 2 solution for 1 to 2 minutes.Surface sterilized root nodules were crushed in sterilized distilled water.Following Serial dilution, bacterial suspension was streaked onto Cango Red -Yeast Extract Mannitol Agar (CRYEMA) plates and incubated at 28±2 0 C for 2 to 3 days (Vincent, 1970).Rhizobial colonies were picked up and sub cultured on Yeast Extract Mannitol Agar (YEMA).

Cultural characters, staining and morphology:-
The cultural characters such as colony shape, colony size, elevation, pigmentation, margin, opacity, mucocity etc. were studied by using standard Microbiology protocols.Bacterial cultures were examined for Gram staining reaction by the method of Hucker and Conn, (1923) using 48 hrs.old cultures.Bacterium shape was observed under the microscope.Cell size was measured by using ocular and stage micrometer as well as photographic methods.

Carbohydrate nutrition:-
The test was carried out on 'non saline' and modified 'saline' Basal medium (Bishop et. al 1976).Different carbohydrates (galactose, glucose, sucrose, dextrose, fructose, maltose, lactose, mannitol and raffinose) were substituted for Mannitol.Every carbohydrate source was added at 1.0% (wt/vol) concentration.Bacterial growth on YEMA slants was removed with cotton swabs and suspended in sterile distilled water to a density 10 -7 per ml. 10 fold diluted cell suspension was added to the wells of a inoculators plate and incubated on to the surface of carbohydrate contained plates.Bishop's agar plate without carbohydrate was served as 'control' the agar plates were incubated for a week at 28 0 C.

Amino acid nutrition:-
To determine utilization of nitrogen, different amino acids such as Amino-N-butyric acid, alanine, aspergin, glycine, histidine, lysine, serine, tyrosine and valine were incorporated in non saline and saline YEM broth at 0.01% concentration.0.1 µl bacterial cultures were added to each test tube.Broth without Yeast extract served as control.The bacterial growth was recorded by using (Shimadzu UV-1800) Spectrophotometer at 600nm after 48 hr.incubation period at 28± 2 0 C.

Results :-
Authentication test:-All strains inoculated on saline and non saline YEMA amended with Cango red were remained creamy.No strain absorbed Cango red.All strains were failed to grow on GPA medium.Rhizobial strains inoculated on Hofer's alkaline medium were unable to grow at high pH (11.00).Isolates were unable to produce 3-Ketolactase because no yellow ring was observed around the colony when flooded with Benedict's reagent on Lactose Agar medium.Cultural characters, staining and morphology:-Colony characters: All isolates were observed for cultural characteristics on saline and non saline YEMA after 48 hrs incubation at 28 0 C. Colonies of all isolates on both the media were circular in shape and creamy in colour.All isolates on both media showed convex elevation, entire margins, mucoid consistency and opaque nature.Comparatively smaller colonies (1 to 3 mm diameter) were observed on saline YEMA while on non saline medium colonies ranged 2 to 6.5 mm in diameter.From above observations it was clear that these were fast growing Rhizobia.

Cell characteristics:-
All isolates were gram negative, rod shaped and non spore forming bacteria.The cell size ranged 0.5 to 1X 1.5 to 2.9 µm on non saline medium while 0.7 to 1.2X 1.7 to 2.9 µm on saline medium.

Salt tolerance:-
Sensitivity of these 31 isolates to NaCl ranged from 1 to 8%.Among the 31 isolates 3 isolates were resistant to 8% concentration of NaCl.From Table No Biochemical and physiological characterization:-Reaction in YEMA: saline and non saline YEMA plates containing Bromothymol blue (BTB) were inoculated with Rhizobium impregnated paper disc showed yellow coloured zone around the disc.It indicated change in the pH of the medium was due to acid producers i. e. Fast growing Rhizobium.All isolates were acid producers and not affected by salinity.Reaction in Litmus Milk: Isolates CPR-11, CVR-7 and CRR-4 showed acidic reaction in saline and non saline litmus milk medium.Amylase activity: A clear halos in the midst of the dark plate was observed in isolate CRR-4 in both saline and non saline starch agar when iodine reagent is applied.Catalase test: All isolates showed positive Catalase activity.Air bubbles produced on the glass slide when H 2 O 2 added to the smear of bacteria.Gelatinase activity: All isolates from saline and non saline nutrient gelatin medium showed liquefaction of gelatin.Nitrate reductase activity: All isolates from saline and non saline medium were reduced Nitrate.Oxidase activity: All isolates from saline and non saline medium showed positive results for oxidase test.Lipid hydrolysis: All isolates in both saline and non saline medium showed a clear halo surrounding the bacterial growth identifies the presence of lipase.Lipase production was not affected by salinity.Citrate Utilization: All isolates were grown on non saline Simmon's citrate agar medium and gave positive test by changing colour from green to blue.Isolates from saline medium remained green due to trace or no growth of Rhizobium isolate.H 2 S production: No black precipitation was observed on Triple Sugar Iron (TSI) medium.Isolates produced gas only.Better results were found on non saline medium than saline medium.Indole test: Bacteria were unable to degrade tryptophan (amino acid).All isolates from modified saline and non saline Tryptophan broth showed negative Indole test.Methyl Red and Vogas Proskaure test (MR-VP): Both tests on saline and non saline MR-VP (Glucose Phosphate broth) were found negative.Physiological and Biochemical characteristics of Rhizobia are given in Table No. 2.

Crystal violet sensitivity:-
All isolates from saline and non saline media were sensitive to crystal violet having 1:1000; 1: 10000 and 1:50000 concentrations.CPR-11 and CVR-7 from both saline and non saline media grown on 1:100000 concentrations.While CRR-4 was unable to grow on both media at 1:100000 concentrations.All isolates from both the media tolerated 1:200000 concentrations.From, Table No. 3, it is proved that fast growing Rhizobia were less sensitive to high concentrations of Crystal violet .
1821 Amino acid nutrition:-Amino acids such as amino-N-butyric acid, alanine, aspergin, histidine, lysine, serine and tyrosine were utilized by all rhizobial isolates from both saline and non saline medium.Glycine and valine were not utilized by all rhizobial isolates.(Table No. 5).

Discussion:-
Results of authentication tests proved that all isolates were Rhizobium and different from Agrobacterium or other bacteria.Colonies obtained from non saline and modified saline media were circular, convex, opaque, mucoid with entire margin.The results were in agreement with Gauri et al., (2011).Comparatively smaller colonies were observed on saline medium.According to Steinborn and Roughly, (1975) and Singleton et al., (1982) salinity increases the generation time of Rhizobia, which results in formation of smaller colonies on saline YEMA.All isolates from non saline and modified saline media were gram negative, rod shaped and non spore forming.The results are similar with Sadowsky et al., (1983).There was a slight variation in size of cell under stressed condition.Rhizobia respond to stress by changing their size and morphology.
It was observed that, sensitivity of these 31 isolates to NaCl ranged from 1 to 10% NaCl.Among the 31 isolates six isolates were resistant to 8% concentration of NaCl.There are many workers who have also recorded sensitivity of Rhizobia to various concentrations of NaCl.According to Kucuk et al., (2006) Rhizobium isolated from root nodules of Phaseolus vulgaris tolerated 5% NaCl.Sharma et al., (2013) reported that Rhizobia isolated from root nodules of Sesbania sesban, Lablab purpureus and Cajanus cajan shown growth on medium containing 40 dsm -1 of NaCl.According to Mandal, (2014) out of 27 strains of Rhizobium trifolli isolated from root nodules of Trifolium alexandrinum tolerated 3% concentration of NaCl.Ali et al., (2009) reported that Rhizobia isolated from Leucaena leucocephala, Tephrosia purpurea and Crotalaria medicagina tolerated 4.5% concentration of NaCl.Bajekal (1996) found that, out of 16 strains of halotolerant rhizobia, two tolerated 3.5% salt, one strain 4.5, two strains, 5.0, four strains, 5.5, two strains, 6.0, three strains, 6.5 and two strains tolerated up to 7.0% NaCl.Rhizobium is more tolerant to salts than their host legume; hence survive in saline condition (Subha Rao et al., 1972 and1974).El-Mokadem et al., (1991) concluded that, salt tolerant Rhizobia can improve yield of legumes under salt condition.Hashem et al., (1998); Shamseldin and Werner, (2005) found that, legume plants grew and survived well in saline condition when they were inoculated with salt tolerant Rhizobia.According to Mandal, (2014) salt tolerant strains of Rhizobium trifolli can be useful under stress condition.
On the basis of biochemical tests; isolate showed acidic to strongly acidic reaction in litmus milk like the previous results of Sadowsky et al., (1983).A yellow ring or colouration around the culture disc on YEMA amended with Bromothymol blue indicated isolates were acid producing or fast growing root nodulating bacteria Rhizobia.The results were in close agreement with Norris (1965).Positive tests were found for Catalase, gelatinase, oxidase and nitrate reductase from non saline and saline condition.A lipid hydrolysis test was also found positive.These results were similar with Sadowsky et al., (1983)

Conclusion:-
Efforts were carried out to develop salt tolerant Rhizobia.The fast growing strains of Rhizobium are alkali tolerant.Rhizobia are more tolerant to alkalinity than their legume host.Hence these Rhizobia improve the alkali tolerance of legumes and become helpful in the amelioration of alkaline soil.
Shahazad et al., (2012), (2014).According to De oliveria et al.,(2007)Rhizobium utilized starch from different sources.Most of the Rhizobia were unable to produce enzyme gelatinase.Hunter et al., (2007)observed negative gelatinase activity.According toSadowsky et al., (1983)fast growing Rhizobia isolated from soybean root nodules could produce enzyme gelatinase but slow growers were unable to produce gelatinase.These tests were not affected by salinity.All isolates utilized citrate from non saline medium but were unable to utilize from modified Simmon's Citrate Agar.According toGauri et al., (2011)all isolates of R. trifolii were unable to utilize citrate.Isolates found negative for Methyl Red (MR), Voges Proskauer (VP) and Indole tests.These findings were closely in agreement withShahazad et al., (2012)characterized Rhizobium strain from root nodules of Alfalfa.From the above observations these Rhizobia were fast growing Rhizobia which are more salt tolerant.

Table No . 2
:-Physiological and Biochemical characteristics of Rhizobia.