Zent ra lbl. :Mikrobiol. 142 (1987), 211-214VEB Gustav Fisch er Verlag J ena

[B ot any Dep artment, Women's College , Ain Sh ams Universit y, Cairo , E gypt ]

Salt and p H Tolerance of Rhizobiam. lequminosarum. TAL 271

F . A. HELEMISH and S. M. A. EL-GA~ll\IAL

Summary

E ffect of pH , ch lorides of sod ium and ca lcium , carbonat e an d bicarbonate of sodium as wellas m agnesium sulphate on growth of R . lequminosaruni TAL 271 was studied. This strain couldgr ow within a wide range of pH an d to lerate both moderat e ac idity and alkalinity with op timumpH of 5.5. It cou ld tolerate Na.Cl Ievels up to 2 per ccnt, while CaC12 was foun d morc toxic. Magne­sium sulpha t e was found to be beneficial for this strain, particu lar ly a t lo wer concentrations.

T he threshold of tolerance for ca rbonat e and bicarbonat e was much lower than in case ofch lor ides . R hiz obiu m could tolerate up to 0.5 per cent bi carbonat e and 0.2 per cent carbon a t e.

Zusammenfassung

E s wu rd e de l' E influt3 von \Vnssers t offioncnkon zen t rat ion , Natrium- und Calciumchlorid,Na triumka rbon a t und -bikarbona t sowio Magnesiumsulfat auf das Wac hstum vo n Rhizobiumlegu mino8arum TAL i il untcrsucht., Di esel' Stamm konnte innerhalb eines weiten p H -Bereicheswaehsen und tolerie rte sowohl mii13ige Azidit iit als auch Alkalinitat bei einem p H -Op t im umvo n 5,5. Er vert rug NaCI-Gehalt e b is zu 2 %' CaC~ hingegen wirkte toxisohe r. 1\fgSO! erwiessieh a ls vo rteilhaft, bcsonders in ni edrigen K on zentrati on en.

Die 'I'oler anzschwelle fu r K arbona t und Bikarbonat la g vi el ni ed riger als Iur Chloride. Del'gepr iift e Stam m vertrug bis zu 0,5 % Bikarbonat und his zu 0,2 % K a rbona t.

Saline, alkaline soils are wide-spread in Egypt , especially in reclam ation zonesTh ese soils are generally not su itable for cult ivating food and fodder legumes. Th edominant ions in soils are sodium, calcium, magnesium, chloride, sulphate , bicarbo nateand carbonate. Most common ar e sodi um and calcium chloride and sulphat e typeof salinity. Alkali soils are developed as a result of t he predominance of carbona t eand bicarbonat es.

GUAHAl\1 and PARKER (1964) found 2 % salinity lethal for most Rhizobium spp. ,although strains resistance t o even 3.0 % NaCI have been found (PALLAI and SEN1966, YADAV and VYAS 197:3, and SUBBA RAO et al. 1972). A facultative halo-tolerantstrain of Rhizooiura. meliloti, t olerating NaCI concentrations as high as 75 111M, wasalso st udied by SAWAGE et a l, (1983). ABDEL WAHAB and ZAHUAN (1979) foundcert ain stra ins of B . lequminosarum. t hat tolera te a concentra t ion of 3.4 % NaCl.The response of R. lequminosarum. 'f AL 271 t o chlorine an d sodium ions was studiedin our laboratory by EL-GA:\D1AL and HELE:\lISH (1984). Fo r rhizobia, opt imump H is ne ut ral or slightly alka line, but t hey are differently sensitive to acidity (ALLENand ALLEN 1950). YADAV and VYAS (1971, 1973) st udied in det ail the response ofroot nodule bacteria to saline, alkaline, and acid cond itions. The activit y of rhizobiato t olera t e salinity, higher pH values, and associated alka lin ity st ress in laborat orymedi um Illay be of predicta ble value in selecti ng st ra ins that form effect ive symbiosiswit h host plan t s in alkaline saline soil. As broad bean is one of t he importan t legu mecrops in Egypt and several tropical countries of Afr ica, direct t est on salt and pH

212 F. A. HELEMISH and S. M. A. EL-GAMMAL

tolerance of it was therefore desirable. The present investigation summarizes ourobservations on the effect of different salts, namely Na2C03, NaHC03, MgS04,

CaCI2, and sodium chloride and pH on growth of a strain of Rhizobium lequminosarum,

Material and Methods

Organism

The strain used throughout this investigation is Rhizobium leguminosarum TAL 271, kindlyprovided from the Microbiology Research Center, Ministry of Agriculture, Cairo.

Broth culturesYeast extract-mannitol medium (ALLEN 1957) was used. Flasks containing 50 ml of liquid

medium were inoculated with 2 ml of bacterial suspension, prepared from scrapings from 2-d-oldagar cultures; the flasks were shaken at the rate of 120 rev. mirr"! at 30°C. Growth in liquidmedium was assessed turbidometrically, using Bauch and Lomb Spectronic 20 at 540 nm every24 h over one week. All treatments were replicated twice.

Effect of pHThe effect of pH on the growth of the bacteria was assessed in liquid medium with the post­

sterilization pH values, viz. 1.5, 3.5, 5.5, 6.5, 7.5, 8.5, and 10. The pH was adjusted by TacusselpH meter.

Effect of different saltsThe salts tested were NaCl, CaCI2, MgS04, sodium carbonate and bicarbonate; each salt except

carbonate and bicarbonate was supplied at the following concentrations, namely: 0.0, 0.2, 0.5,0.8, 1, 1.5, 2.0, 2.5, and 3 % (wjv), respectively. Carbonate and bicarbonate was added to thegrowth medium in concentrations of 0.0,0.1,0.2,0.5,0.8, and 1 % (wjv). All other growth condi­tions were performed as previously mentioned.

Results and Discussion

The response of R.leguminosarum TAL 271 to different salts and pH is summarizedin Table 1 and 2.

Table 1. Optical density after 96 h, when R.leguminosarum TAL 271 was grown in broth of differingconcentrations of salts and pH

Salts Concentrations (%)

0.0 0.2 0.5 0.8 1.0 1.5 2.0 2.5 3.0

NaCI 0.62 0.56 0.53 0.84 0.82 0.78 0.64 0.61 0.60CaCl 2 0.94 1.05 1.05 1.06 0.88 0.91 0.84 0.78 0.53MgS04 0.56 0.76 0.84 0.88 0.94 0.92 0.90 0.86 0.84

Concentrations (%)

0.0 0.1 0.2 0.5 0.8 1.0

Na2C03 0.88 1.02 0.90 0043 0040 0.35NaHC0 3 0.88 1.14 1.22 1.19 0.87 0.84

pH values

1.5 3.5 5.5 6.5 7.5 8.5 10

pH 0.14 0.90 2.90 2.88 2.40 2.32 0.65

Salt and pH Tolerance 213

Table 2. The effect of salts and pH on R. lequminosarum. TAL 271

Response Concentrations (%) ofto salts and pH

NaCl NaHC03 Na2CO, CaC12 MgS04 pH

Optimum 0.8 0.2 0.1 0.8 1.0 5.5

Tolerant 2.0 0.8 0.5 i.s 3.0 8.5

Sensitive 2.5 1.0 1.0 2.0 3.5

Inhibited 1.5

The results showed that this strain of R. lequminosarum. tolerates both highsalt concentrations and a wide range of pH. Tolerance of this strain to both acidityand moderate alkalinity is in agreement with the result reported by YADAV andYYAS (1973), OKAFOR and ALEXANDER (1975), who found that some strains of cowpeagroup rhizobia could grow at pH values as low as 3.5. Contrary to YADAV and VYAS(1971), a pH of 10 was found to be critical to our strain. They showed also that dif­ferent rhizobia strains varied widely in their sensitivity to pH.

With respect to NaCl tolerance, this strain of R. lequminosarum. showed increasein growth with an increase in NaCl up to 2 % concentration (Table 2). The literatureshows a wide variability in NaCI tolerance of rhizobia. While STEINBORN and ROUGH­LEY (1975) reported that R. lequminosaruni was tolerant up to 1 % NaCl, ABDELWAHAB and ZAHRAN (1979) found that R. meliloti and R. lequminosoruni were ableto tolerate high salt concentrations and could grow at 3.4 % NaCl concentration.In previous investigations (EL-GAMMAL and HELEMISH 1984) this strain was foundto tolerate NaCl salinity much more better in broth culture than in peat culture.

Unexpectedly, addition of NaHC03 or Na2C03 to the growth medium of R. le­quminosarum. increased the growth-rate at concentrations up to 0.5 per cent NaHC03and 0.2% Na2C03 (Table 1). Sodium carbonate was found to be more toxic thanthe carbonate. According to YADAV and VYAS (1971) a concentration of 0.2-0.8and 0.6-0.8 per cent bicarbonate was lethal for Crotalaria juncea and Glycine maxstrains, respectively. RAO et al. (1974) found that 0.2 % NaHC03 and 0.1 % car­bonate concentrations reduced growth of R. lequminosarum. 1007 by more than 50%.

MgS04 ions were found to be beneficial for the growth of this strain at lowerconcentrations (Table 2), thus 1.0 per cent concentration was optimum for growth.It was also clear that R. lequminosarum was stable at 3.0 per cent concentration,and no inhibition of growth compared with the control was observed. This resultis in agreement with YADAV and VYAS (1971a) who found the same effect of MgS04on the growth of Glycine max strain.

Calcium chloride was optimum at 0.8 % and inhibited markedly the growth rateat a concentration of more than 1.5 % (Table 2). The toxicity of calcium chloride washigher than that of NaCl, carbonate, and bicarbonate. This is in agreement withSTEINBORN and ROl;GHLEY (1975) who found that calcium chloride was more toxicthan sodium chloride in broth and peat culture. Preparation of soil inoculum fromthis strain is therefore desirable which would be potentially better able to withstandthe condition of reclamation zones. Research for new compounds which enable rhizobiato overcome salinity was also another way to solve salinity problem. SAWAGE et al.(1983) found that addition of 10 mM glycine betaine improve the salt tolerance ofRhizobium meliloti. RAo et al. (1974) found that the degree of salinity/alkalinityconducive for good nodulation was definitely different from the limit of toleranceof Rhizobium and the host to the respective salts. However, the limits of salt tolerance

214 I". A. HELEMISH and S. M. A. EL-GAlIIMAL, Salt and pH Toleran ce

of the legume as well as the limits of salt tolerance of the bacterium for successfulnodulation and symbiosis are also important parameters involved in salinity problemand need further study.

References

ABDEL 'W ARAB, and ZAHRAN, H. H .: Sal t tolerance of Rhizobium spec ies in broth cult ure . Z.a llg. Mikrobiol. 19 (1978), 681-685.

ALLEN, O. N.: Experiments in soil Bact eriology. 3rd ed. Minneapolis 1957.ALLEN, E. K., and ALLEK, O. N. : Bio chemi cal and symbioti c properties of rhizobia. Bact. R ev.

19 (1950), 273-330.EL -GAMlIIAL, S. :\f. A., and HELEMISH, F. A. : Salt tolerance of Rhizobium legumino surum in peat

an d broth cultures. Academi c J ournal of Science. Girl's College, Dammam, Saudi-Arabia(1984) (under press).

GRAHAM, P. H., and PARKER, C. A.: Diagnostic features in the charact erization of root nodulebact eria of legumes. PI. Soil 20 (1964), 383-396.

OKAFOR, N., and ALEXANDER, 1\1. : Preliminary physiological st udies on cowpea rhizobia. SoilBioI. Biochem. 7 (1975), 405-406.

PALLAI,R. N., and SEN, A.: Salt tolerance of R. trifolii. Indian J. Agri c. Sci. 36 (1966),80-84.RAo, N. S., KUMARI, M. L., SINGH, C. S., and BISWAS, A.: Salinity and alkalinit y in relation to

legume Rhizobium symbiosis. Proo, Ind. Natl. Sci. 40 (1974), 544-547.SAWAGE, D., HAMELINY, and LARTIER, Glycine betaine and other st ruc t ura lly related com­

pounds improve the salt tolerance of Rhizobium meliloti pl an t. Sci. Lett. 31 (213) (1983),291-302.

SUBBA RAO, N. S., LAKSHlIII-K uMARI, 1\1., Srxcn, C. S., and 1\IAGT, S. P.: Nodulation of lucerne(Medi cago sa t iva L.) under the influen ce of sodium chloride. Ind. J. Agri c. Sci. 42 (1972),384-386.

STEINBORN, J., and ROUGHLEY, R. J. : Toxici ty of sodium an d chloride ions to Rhizobium spp.in broth an d peat cult ures . .J, Appl. Bacteriol. 39 (1975), 133-138.

YADAV, N. K., and VYAS, S. R.: Note on the response of ro ot-nodule rhizobi a to saline, alkaline,an d a cid conditions. Ind. J. Agrio, Sci. 41 (1971), 1123-1125.

YADAV, N. R., an d VYAS, S. R. : Effect of salts and pH on the growt h of Rhizobium strains. Ind.J. Mierobiol. 11 (1971 a), 97-102.

YADAV, N. K., and VYAS, S. R.: Salt and pH Tolerance of Rhizobia. Folia 1\Iicrobiol., Praha18 (1973) , 242-247.

Author's ad dress :

Dr. F. A. HELElIIISH, Botany Department, Women's College, Ain Shams University, Cairo,Egypt.