Many physiological aspects of Rhizobium-legume symbiosis are still poorly understood, although rhizobial root nodules of leguminous plants have created great interest among scientists for a long period of time. The production of rhizobial extracellular polysaccharides (EPS) is one of these aspects. Rhizobial EPS was shown to be involved in the Rhizobium-legume symbiosis. Olivares et al. (1984) reported about the enhancement of nodulation by EPS. Rhizobium species were described which were able to produced EPS also in culture (De and Basu, 1996; Ghosh et al., 2005). Skorupska et al. (2006) also reported that extracellular polysaccharides may be involved in invasion and nodule development, bacterial release from infection threads, bacteroid development, suppression of plant defense response and protection against plant antimicrobial compounds.
Sesbania sesban (L.) Merr. is a widely cultivated green manure crop in Andhra Pradesh, India. Very little information about the EPS production by the symbiont of S. sesban. The objective of this study was to screen the maximum EPS producing strain from S. sesban and also to increase the production of EPS through optimization of cultural conditions of the strain, which produced maximum amount of EPS.
MATERIALS AND METHODS
Microorganism, Medium and Growth Conditions
Twenty six Rhizobium strains were isolated from fresh healthy root
nodules of Sesbania sesban collected from different regions of Andhra
Pradesh, India. The study was conducted in December, 2006 in the Department
of Microbiology, Acharya Nagarjuna University. The basal medium for the bacterial
growth and EPS production were the yeast extract mineral medium (Skerman, 1959) with 1% mannitol. The strains were incubated in 25 mL of the medium in 100 mL conical flasks in three replicates at 30±2°C for 48 h (optimum time for maximum EPS production). The growth was measured spectrophotometrically at 540 nm.
Production of EPS on Different Sources
Different carbon sources were added separately to the basal medium replacing
mannitol. Individual effect of different chemicals with most suitable carbon
source on EPS production was tested. For maximum EPS production by the strain
the medium was enriched with different supplements which individually increase
the EPS production to maximum level. All the supplements added to the medium
were filter sterilized.
Isolation of EPS
Isolation of EPS was done by following the method described by Dudman (1976)
and collected by centrifugation, dissolved in minimum volume of distilled water,
reprecipitated with 3 volumes of acetone, centrifuged, dialyzed and lyophilized.
For identification of sugar monomers, dry EPS was hydrolyzed in a sealed tube
with 0.5 M BaCO3 and concentrated at 45°C under reduced pressure.
EPS was chromatographed on Whatman No. 1 paper using butanol: acetic acid: water
(4:3:1) as solvent system. Spraying reagent used for identification of sugar
components was aniline phthalate (Partridge, 1949). For Gas Liquid Chromatography
(GLC), sugar derivatives (paracetylated alcohols) were prepared from dry lyophilized
polysaccharides (Ghosh et al., 2005) and injected into GLC apparatus.
The sugar derivatives were identified by comparisons of their retention times
with those of authentic standards.
Estimation of EPS
The dialyzed cell free supernatant was used for EPS estimation by phenol-sulphuric
acid method following Dubois et al. (1956). Uronic acid estimation in
the EPS was performed by Carbazole reaction (Dische, 1947).
The data were statistically analyzed using correlation coefficient between
growth and EPS production.
RESULTS AND DISCUSSION
The Rhizobium strains isolated from root nodules of S. sesban were designated as Rhizobium SS1 to SS26. The isolated strains were identified as species of Rhizobium following Bergeys Manual of Systematic Bacteriology (Jordan, 1984) and plant infection test (Vincent, 1970). The Rhizobium strains were fast growers and reached stationary phase at 48 h. Among the 26 Rhizobium strains tested, the Rhizobium SS5 produced maximum amount of EPS on yeast extract mannitol medium (Table 1). Maximum EPS production was also observed at 48 h by this strain. As Rhizobium SS5 produced more amounts of EPS, further tests were carried out on this strain.
All the fourteen carbon sources (1%) promoted both growth and EPS production,
but maximum amount was observed in galactose followed by mannitol (Table
2). But the mannitol was the best carbon source was reported earlier in
Rhizobium D110 sp. from Dalbergia lanceolaria (Ghosh et al.,
2005). The optimum concentration of galactose required for EPS production was
found to be 2.0% (Table 4).
|| Production of Extracellular polysaccharides (EPS) by Rhizobium
strains from Sesbania sesban
|*: Correlation coefficient between growth and extracellular
polysaccharide production (r = 0.71)
|| Effect of different carbon sources on growth and extracellular
polysaccharide production by Rhizobium SS5
|*: Control was devoid of carbon source; Correlation coefficient
between growth and extracellular polysaccharide production (r = 0.67)
Among the nitrogen sources tested, maximum EPS production was observed in sodium nitrate followed by potassium nitrate (Table 3). The optimum concentration was found to be 0.1% (Table 4). But, potassium nitrate at 0.1% concentration increase EPS production in Rhizobium D110 strain from Dalbergia lanceolaria (Ghosh et al., 2005).
Among the different vitamin sources tested, Ca-pantothenate was most effective
source for maximum EPS production at 1 μg mL-1 (Table
3). But, D-Biotin at 1 μg mL-1 increased both growth and
EPS production were reported in Azorhizobium caulinodans from Aeschynomene
aspera (Ghosh and Basu, 2001).
||Effect of different nitrogen sources and vitamins on growth
and extracellular polysaccharide production by Rhizobium SS5
|*: Control was devoid of any type of additional nitrogen and
vitamin sources; Correlation coefficient between growth and extracellular
polysaccharide production in nitrogen sources (r = 0.99), in vitamins (r
||Effect of different concentrations of galactose and sodium
nitrate on growth and extracellular polysaccharide production
||Increase in growth and extracellular polysaccharide production
by Rhizobium SS5 using most effective supplements
|*: In the control, bacteria were grown on yeast extract mannitol
medium. In other cases the medium was supplemented with galactose (2%),
Ca-pantothenate (1 μg mL-1) and sodium nitrate (0.1%)
To test the maximum EPS production by Rhizobium SS5 strain in culture,
the supplements which individually increased the production to the greater extent
was added to the medium. The strain which initially produced 3900 μg mL-1
EPS in basal YEM medium was induced to yield more amounts of EPS through optimization
of cultural conditions (Table 5). The EPS produced by this
strain contained galactose, glucose, xylose, rhamnose and raffinose, which were
identified by paper and gas liquid chromatography. The sugar isomers contained
30.2% galactose, 20.9% glucose, 20.4% xylose, 16.3% raffinose and 12.2% rhamnose,
taking total of the five sugars as 100% (Table 6).
|| Relative (%) of sugar monomers in the extracellular polysaccharides
of Rhizobium SS5 as identified by GLC
Hollingsworth et al. (1985) also observed the presence of galactose, glucose and mannose in EPS, which were secreted by Rhizobium strain of M1-50A, M6-78 and IRC 253 of cowpea rhizobia. EPS of some members of Rhizobiaceae contains mannitol and fructose (Breedveld et al., 1993). These have indicated that there are variations in the sugar monomers from different Rhizobium spp.
The EPS secreted by Rhizobium SS5 was acidic, indicating the presence of uronic acid. The amount of uronic acid was found to be 352.9 μg mL-1 of EPS. Amemura et al. (1983) have reported that most extracellular acidic polysaccharides of Rhizobium trifolii contained D-glucuronic acid.
Correlation between the growth and EPS production in YEM medium is positive (r = 0.71). The effect of carbon, nitrogen and vitamin sources also showed positive correlation that of nitrogen sources is highly positive (r = 0.99).
All the supplements which increased the EPS production in culture could be available for the Rhizobium SS5 in the soil from the plant as root leachate. This might stimulate the Rhizobium to produce more polysaccharides helping to promote the infection and enhance nodulation of legumes (Ghosh et al., 2005). Moreover, the increased EPS production by the strain SS5 could be useful for the industry. Glycan, dextran and xanthan of bacterial origin are of commercial importance.
We thank Andhra Pradesh Council of Science and Technology (APCOST), Hyderabad, India, for financial assistance in the form of Young Scientist Fellowship (YSF) to MS.