' P-Isolation and Characterization of Salt Tolerant Strains of Sinorltizobium meliloti •

Twenty four strains of Sinorhizobium meliloti were isolated from root nodules of alfalfa plants. Host plants collected from different agroclimatic regions of Ninavah province-Iraq. Five strains, vis. FA7, FA8, FAlO, FAll and FA12 were found tolerant to 6% NaCI. All the five strains were able to grow on rhizobia! minimal medium. The five studied strains showed multiple antibiotic resistance. The more sufficient production in shoot group in alfalfa plants found when the plants inoculated with F A8 and FA 7 strains. pH changes in MSY solid medium study revealed that changes in pH towards acidity. Reduction in colony forming units (CFU) was observed under salt conditions in comparison with normal conditions. Production of cell surface molecules in these five strains , such as, P-(1 ----+ 3) glucans, cellulose fibrils and exopolysaccharides were also studied. Heat curing of Sym plasmids study revealed that at least one of the salt tolerant genes lays on Sym plasmids. •Presented at the first conference on Biology , University of Mosul ,college of Education , 4-5 September 2007


ABSTRACT
Twenty four strains of Sinorhizobium meliloti were isolated from root nodules of alfalfa plants. Host plants collected from different agroclimatic regions of Ninavah province-Iraq. Five strains, vis. FA7, FA8, FAlO, FAll and FA12 were found tolerant to 6% NaCI. All the five strains were able to grow on rhizobia! minimal medium. The five studied strains showed multiple antibiotic resistance. The more sufficient production in shoot group in alfalfa plants found when the plants inoculated with F A8 and FA 7 strains. pH changes in MSY solid medium study revealed that changes in pH towards acidity. Reduction in colony forming units (CFU) was observed under salt conditions in comparison with normal conditions. Production of cell surface molecules in these five strains , such as, P-(1 ----+ 3) glucans, cellulose fibrils and exopolysaccharides were also studied. Heat curing of Sym plasmids study revealed that at least one of the salt tolerant genes lays on Sym plasmids.

Introduction
Atmospheric N 2 fixed symbiotically by the association between Rhizobium species and legumes represents a renewable source of N for agriculture (1). Soil salinity is a major limiting nitrogen fixation factor because of its adverse effects on the growth of the host plant, on its root nodule bacteria and on symbiotic development (2). Rhizobia vary in efficacy of osmoregulation-response to salt stress (3,4,5). This capacity of adaptation of the microbial population was exhibited in a study of the survival of Bradyrhizobiumjaponicum in sludge-amended soils (6). Some salinity tolerant rhizobia were reported which showed better nodulation and nitrogen fixation than salinity sensitive rhizobia under saline conditions (7). Though these salt tolerant strains seem to accumulate osmoprotectants such as glycine betaine, proline betaine glutamic acid etc., the biochemical mechanisms of salt tolerance is not fully understood (5).
Several cell surface molecules like exopolysaccharides and ~-(1 --.2) cyclic glucans, which play a role in early stages of infection process also play a role in stress tolerance. ~-(1 -+ 2) glucan has been found to play a major role in osmotic adaptation (8). Nogales et al. (9) identified eight gene loci required for adaptation to high external NaCl. Seven different genes involved in salt tolerance were isolated and characterized from S. meliloti mutants (1 0). Miller-Williams et a!. ( 11) studied the determinants necessary for adaptation to high NaCl concentrations and they isolated salt-sensitive mutants of s. meliloti strain Rml 021. Payakapong eta!. (12) identified two clusters of genes involved in salt tolerance in Sinorhizobium sp. strain BL3. Bacem et al. (13) isolated salt tolerant rhizobia from a Tunisian oasis that are highly effective for symbiotic N 2 fixation.
Selection of salt tolerant and efficient strains is very useful as the selected strains can be introduced into soils of respective stress where competition from naturally occurring rhizobia is lacking. Keeping these observations in mind, the present work was taken for collection of a number of strains of rhizobia from different agroclimatic regions of Ninavah State/ Iraq, selection of stress tolerant strains in the laboratory as well as to characterize these strains for their symbiotic performance and other features.

Bacterial strains
Twenty four strains of Sinorhizobium meliloti were isolated from root nodules of alfalfa plants. The plants were collected from different agroclimatic locations ofNinavah State/ Iraq.

Host plant cultivar
The seeds of alfalfa were obtained from local market.

Maintenance of rhizobia/ strains
Purified isolated strains were streaked on slants of MSY solid medium. After a growth period of 24-48 hours at 28±2 °C, slants were stored at 4 oc in a refrigerator. Reculturing of rhizobia} strains was done each two months.

.1. Antibiotics
Five antibiotics with four concentrations, viz., 100, 200, 300 and 400 Jlg/ml were used in this study. Stock solutions of tetracycline hydrochloride (Tc), chloramphenicol (Cm) and ampicillin (Am) were prepared in ethanol, while streptomycin sulphate (Sm) solution was prepared in distilled water. Nalidixic acid (Nal) was dissolved in o.o5 N sodium hydroxide. Antibiotic solutions were sterilized by passing them through 0.45Jlm membrane filters and stored at 4 °C. Different concentrations of antibiotic were added to the autoclaved medium after cooling it to 50 °C, just before plating (20).

Salt
Sodium chloride was added to the medium before autoclaving. Percentage concentration method (%, w/v) was followed for supplementing this salt into the medium.

Dyes
Aniline blue for cyclic ~-( 1-+ 3) glucans production or calcoflour white for testing succinylated exopolysaccharides was added to the YEM medium at the rate 0.02 o/o (w/v), while congo red for testing cellulose fibrils production was added to the same medium at 0.1 mg/ml final concentration. Each of these reagents was added to the medium before autoclaving ( 1 7).

pH indicator dye
Bromothymol blue dye was added to the MSY solid medium for the pH changes study at the rate of 2.5 mg/ 100 ml medium before autoclaving (20).

Isolation of rhizobia/ strains from their host plant
Vincent ( 15) procedure was followed for isolating rhizobia! strains from the root nodules. Three to four pinkish nodules were washed in distilled water and exposed to 95 % (v/v) ethanol for 2-4 minutes. These nodules were then washed in sterile distilled water and immersed in 0.1 % (w/v) acidified HgCh (HgCh, 1.0 gm; cone. HCl 5 ml and water 1.0 liter) for 3-6 minutes. The surface sterilized nodules were washed thoroughly 5-7 times with sterile water to remove traces of mercuric chloride and alcohol. The nodules were then crushed aseptically in 1.0 ml Raad Hassani Sultan & Fa\vz A. AL-Safar sterile saline (0.85 % w/v NaCl) with a sterilized glass rod. Suspension (0.1 ml) was ·spread on MSY solid medium and the plate was incubated at 28±2 oc for 2-4 days. Transparent mucoid or gummy colonies were picked for further purification.

Screening for salt tolerance
The growth of all isolated strains of S. meliloti was tested by streaking these strains on MSY medium containing (0, 2, 4, 6 and 8 %) of NaCl. Incubation was done at 28±2 oc for 4-6 days (21 ).

Plant inoculation studies
Alfalfa seeds were sterilized as described by Vincent (15) and transfetTed onto nitrogen free agar slants in 20 x 2.5 em tubes. Two 2days old seedlings in each tube were inoculated with 10 8 cells (suspended in sterile distilled water) of a particular rhizobia! strain. The growth conditions for the plants were 2000 lux light, a photoperiod of 16 hr, a dark period of 8 hr and 25 °C temperature. The morphological features of plants were recorded six weeks after inoculation. For determining the dry plant shoot weight, the plant tops were collected and dried in an oven at 65 ~C for 72 hr and then weighted. Rei solation of bacteria from nodules was done to confirm the nodule occupancy by a particular strain.

Symbiotic p/asmids heat curing study
To emphasize the position of salt tolerance genes whether it lay on chromosome or on symbiotic plasmids, heat curing of Sym plasmids procedure was done as follows: Approximately 10 10 cells from a log phase culture of salt tolerant strain grown on TY medium were evenly spread on the surface of solid YEM medium. The cultures were incubated at 3 7 °C for 7 days, during which no growth occurred. The YEM plates were placed at room temperature, and after 5 days, about 100 single colonies arose per plate (22). To confirm symbiotic plasmids curing; plant nodulation test, antibiotic sensitivity and 6 % NaCl tolerance were done as reported by Vincent (15), Hussein (20) and Sadowsky eta/. (21).

Isolation of salt stress tolerating rhizobia/ strains
Among studied strains five strains vis. FA7, FA8, FAlO, FAll and FA12 could grow on MSY medium supported with 6% NaCl, 8% K 2 S0 4 and pH 4.5 (data not shown) choosed for further studies. Kumar (23) and Hussein (20) have isolated rhizobia! strains from Indian soils which tolerated 4 and 5 o/o NaCl, respectively. Screening of rhizobia! strains for survival and growth in salt stress laboratory media has resulted in some success in improving the nodulation of some legumes in stressed soils (24). Tolerance of S. meliloti strains to salt stress due to ability of rhizobia! cells to accumulate potassium ions inside the cells (25).

Antibiotic resistance patterns of salt tolerant stress
Results of this study showed that the five S. meliloti strains were sensitive to 100 ~g/ml ofTc whereas two strains (FAll and FA12) were able to grow on MSY medium supplemented with 200 ~g/ml of (Cm), increasing the concentrations of this antibiotic up to 400 ~g/ml resulted in inhibition of growth of all the studied strains. These strains also were resistant to Am up to 400 ~g/ml. FA 10 strain was able to grow with 3 00 ~g/tnl Sm whereas there is no growth with 300 and 400 jlg/ml for all strains. All strains, except FA12 grew on MSY medium supplemented with Nal up to 400 ~g/ml. Multiple antibiotic resistances to antibiotics explain existences of these strains in soil and then enter symbiotic relation with their host plant (6). The antibiotic resistance may be helpful in further genetic analysis of salt tolerance in these strains (20).

Test for growth on Rhizobia/ Minimal Medium (RMM)
Results of this study revealed that the five studied strains were able to grow on RMM solid medium after 4-5 days incubation period. This result revealed that non of these strains suffered from auxotrophy (26). Such these strains are useful for genetic studies ( 12).

Symbiotic characteristics of alfalfa plants inoculated with NaCl tolerant strains
Results of this study showed that the nodules induced by all the studied strains were pinkish in color and were located on both primary and lateral roots. The nodules were cylindrical in shape except in the strain FAll, the induced nodules were branched ( Table 1). The plants inoculated with FA12 strain showed maximum mean shoot lengths (20.5 Cm). Mean number of days for the appearance of first nodule varied from 7.2 in strain FA12 to 10.1 in strain FAll. The mean number of nodules per plant ranged from 3.5 in strain FAIO to 7.2 in strain FA12. The minimum mean nodule dry weight per plant (0.4 mg) was observed in strain FA10, whereas strain FA12 produced the maximum value (2.1 mg) for this character. The mean shoot dry weights per plant ranged from 18.9 in strain FAlO to 44.1 mg in strain FA8. The more sufficient production in shoot group in alfalfa plants was found when the plants were inoculated with FA8 and FA 7 strains, respectively. However, the performance of these ·strains under salt stress conditions in the field remains to be studied (20).

pH changes during growth of the NaCl tolerant strains
On solid MSY medium containing 6% NaCl change to acid pH, as shown by the change of bromothymol blue dye to yellow color, occurred after 48 hours in all S. meliloti strains. Only one salt tolerant strain (FA 7) changed the pH to acidic on solid medium under normal condition.
Payakapong et al. also obtained colonies from salt tolerance strains of Sinorhizobium sp. showed acidic reactions on YEM agar containing bromothymol blue (12). Howieson (27) reported that during the growth of S. meliloti in yeast extract/ sugar preparation, incomplete oxidation of sugars takes place which yields acid end products. But other workers Hernandz and Focht (28); Cadahia et al. (29) found no correlation between growth and pH changes of the medium in case of cowpea and chickpea rhizobia. The pH changes during growth seem to be strain specific(20).

Colony forming units (CFUs) of the NaCI tolerant strains
· · When strains were grown in medium without and with 6 % NaCl, CFU decreased in 6 % NaCl medium in all strains as compared to that under normal condition ( / ml after 72 hr incubation for FA8 strain. Minimum CFU under same condition was 2.9 x 10 9 / ml after 24 hr incubation for FA1 0 strain. Other researchers also showed inhibition in growth under salt condition (12,20).

Production of cell surface molecules under normal and salt stress condition
Under normal condition the ability to produce cyclic P-(1-+ 3) glucans was present in the all studied strains. This ability increased under salt stress condition (6 %) for the strain FA7 and FA12. Also all the studied strains were able to produce cellulose fibrils and the salt stress conditions have no effect on the production of cell surface molecule in any of the strains. The ability of production of succinylated exopolysaccharide (SEPS) was unaffected by salt stress in studied strains except FA1 0, where the production increased under salt stress conditions in comparison with normal condition. Swamynathan and Singh (12) reported that the purine auxotrophy has effect on P-( 1 -+ 3) production.
In this study all the strains were prototroph so the production of these cell surface molecules was normal. Hussein (20) also reported that the salt stress condition has no effect on cellulose fibrils production. Many researchers revealed the role of exopolysaccharide in rhizobium-legume symbiosis especially in initiation and elongation of infection thread when rhizobium invades root hairs of host plants and induce N 2 fixing root nodule (30,31,32). Howieson et al. (24) reported that there is no relation between production of exopolysaccharides and stress condition in S. meliloti, while in Rhizobium spp. the production of exopolysaccharide increased under stress condition (33 (Table 3). Salt stress condition resulted in decreasing the motility values in FA 10 and FA 12 up to one third. Less effect of salt stress was on FA 7 strain in comparison with normal conditions, therefore this strain may be used as inoculants under salt stress conditions. Hussein (20) revealed that salt stress has different effect on motility of S. meliloti salt tolerant strains. Results revealed that heat curing of S. meliloti salt tolerant strains resulted in Nod· phenotype when alfalfa plants inoculated with these strains. Obtained colonies after heat curing of symbiotic plasmids of salt tolerance strains showed sensitivity against Cm, Am, Sm and Nal antibiotics when the MSY solid medium supplemented with minimum concentration (1 00 f.!g/ml) of the mentioned antibiotics. These results mean successful of heat curing of symbiotic plasmids. The loss of Rhizobium nodulation properties strains has been reported from many laboratories (34). Zurkowski (35) isolated many non-nodulating mutants of Rhizobium leguminosarum bv. Trifolii after incubation at an elevated temperature with a high frequency of loss of nodulation properties.
After ~heat curing of S. meliloti salt tolerant strains, growing colonies were recultured on MSY medium supplemented with 6 % NaCI. No visible growth was noticed for the five studied strains. This result revealed that at least one of the salt tolerant genes lay on Symbiotic plasmids. Payakapong et a!. (12) identified two gene clusters for salt tolerance in both fast and slow growing rhizobia.