REVIEW / SYNTHESE

Stress tolerance in Rhizobium and Bradyrhizobium, and nodulation under adverse soil conditions

PETER H. GRAHAM R hizobium Research Laboratory, Department of Soil Science, University of Minnesota, St Paul, MN 551 08, U. S. A.

Received August 22, 1991

Revision received November 18, 199 1

Accepted November 21, 1991

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Introduction 475 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Soil acidity 475

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hydrogen ion concentration 475 Aluminum and manganese toxicities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 477

. Calcium and phosphorus requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 477 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Osmotic stress and salinity 478

Temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 479 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 480

Key words: acidity, temperature, nutritional deficiency, osmotic stress, infection, shock proteins.

Can. J . Microbial. 38, 475-484 (1992)

Introduction Few agriculturalists have the luxury of production under

ideal conditions. For the great majority, and particularly in the third world, stresses of one form or another are an integral part of the crop cycle, and may pose a severe yield constraint. Thus in Latin America alone there are more than 800 million ha of oxisols and ultisols (Cochrane 1979), which are inherently acidic (Kamprath 1984), while current esti- mates of the agricultural land affected by salt range from 400 to 950 million ha (Shannon 1984). Postel (1990) reports more than 60 million ha of irrigated land worldwide in which continued salinization poses a threat to crop yield. Leguminous plants will play an important role in the devel- opment of sustainable agricultural systems for such areas, but the response of particular legumes to stess is not a prop- erty of the host plant alone, but demands consideration of the inoculant rhizobia and the process of symbiosis itself. This review emphasizes .the effects of acidity, osmotic stress, and temperature on Rhizobium and Bradyrhizobium, and on the symbiosis of these organisms with legumes.

Soil acidity Soil acidity affects all aspects of nodulation and nitrogen

fixation from survival and multiplication of the rhizobia in soil, through infection and nodulation, to nitrogen fixation. Hydrogen ion concentration per se, aluminum and manganese toxicities, and deficiencies of calcium, molyb- denum, and phosphorus can all contribute to this problem, with their relative importance different in each soil.

Karanja and Wood 1988a). Fast-growing rhizobia have generally been considered less tolerant to acid pH than bradyrhizobia, but strains of Rhizobium loti and type I1 bean rhizobia (now designated Rhizobium tropici (Martinez- Romero et al. 1991)) have also proved highly acid tolerant, with growth in some cases to pH 4.0 (Cooper 1982; Cunn- ingham and Munns 1984; Wood et al. 1988; Graham et al. 1992). Strains of Rhizobium meliloti are particularly affected by acidity, soil pH being the major factor limiting their num- bers in soil (Brockwell et al. 1991). Brockwell et al. (1991) found an average of 89 000 organisms per gram in soils where the pH was 7.0 or more, but only 37 per gram in soils of pH less than 6.0. Agar plate methodologies (Graham et al. 1982; Ayanaba et al. 1983) have provided a convenient means for the comparison of strain differences in pH tolerance, with isolates having higher than normal tolerance to acidity now identified in most groups of root-nodule bac- teria. Some bradyrhizobia have even been found to grow better at acid pH than at pH 7, and on isolation from nodules need to be cultured in medium of pH 4.5-5.5 (Date and Halliday 1979; Gomez de Souza et al. 1984; Sylvester- Bradley et al. 1988).

Bushby (1990) found that only Bradyrhizobium strains isolated from acid soils had a net positive charge, and postulated a relationship between their surface-charge char- acteristics and the soil from which they came. Despite this, strains isolated from acid soils are not necessarily pH tolerant (International Institute of Tropical Agriculture 1980; Wood and Shepherd 1987; Richardson and Simpson 1988, 1989; Asanuma and Ayanaba 1990), but rather are

Hydrogen ion concentration concentrated in more favorable microsites in the soil. Thus, The lowest pH at which rhizobia will grow varies with for a soil of pH 4.2, only 96 of 481 isolates were capable

cultural conditions, but when the medium pH is adjusted of growth at even pH 4.70 (Richardson and Simpson 1988). after autoclaving and the inoculant density is low, few strains The basis for pH tolerance in neutrophilic species is will grow below pH 4.5 (Date and Halliday 1979; Keyser through the regulation of cytoplasmic pH, with cells main- and Munns 1979a, 1979b; Graham et al. 1982; Cooper 1982; taining an internal pH of 7.5 to 8.0 against a pH differen- Ayanaba et al. 1983; Lowendorf and Alexander 1983; tial with the outside environment of as much as three pH Printed in Canada / lmprime au Canada

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476 CAN. J . MICROBIOL. VOL. 38, 1992

units (Booth 1985). This is usually achieved through the explusion of protons, with uptake of potassium ions needed to balance cell charge. Some species also possess a cyto- plasmic buffering capacity (20-1000 nmol hydrogen ions per pH unit per milligram cell protein (Krulwich et al. 1985)) or the ability to regulate cell metabolism and offset the accu- mulation of acidic end products (Gottwald and Gottschalk 1985; Marquis et al. 1987; Foster and Hall 1990). Several recent studies have also reported "acid-shock" proteins, pro- teins synthesized in greater amount or only detected under acid growth conditions (Taglicht et al. 1987; Bhagwat and Apte 1989; Heyde and Portalier 1990; Hickey and Hirschfield 1990). The nature of these substances and possible similari- ties with other shock proteins remain to be determined.

For the rhizobia, the basis of pH tolerance remains uncer- tain. Cytoplasmic pH has been measured using radioactively labeled weak acids or fluorescent probes, but the results are not clear cut. For an external pH of 6.15-8.45, Gober and Kashket (1984) found the cytoplasmic pH (pHi) of Bradyrhizobium sp. 32H1 to range from pH 7.60 to 8.90 at 20% oxygen and from 6.15 to 8.90 at 0.2% oxygen, with pHi always more alkaline than the external pH. The same authors (Gober and Kashket 1985) found cytoplasmic pH in R. meliloti to vary from pH 7.6 to 8.0, although Tremblay and Miller (1983) found cytoplasmic pH in this organism nearly constant at 6.5 to 6.7. In the acid-tolerant R. meliloti strain WSM419 decline in pHi as the external pH fell from 7.0 to 5.5 was less than 0.3 pH units, but in an acid-sensitive Tn5 mutant derived from it, cytoplasmic pH fell from 7.3 to 6.1 over the same external pH range (O'Hara et al. 1989).

Changes in metabolic activity as a consequence of exter- nal pH are perhaps implicit in the ability of the rhizobia to enhance their external pH prior to, or concomitant with, the initiation of growth in acid medium (Ayanaba et al. 1983; Howieson 1985). Ayanaba et al. (1983) noted signifi- cant ammonia production in acid-tolerant cowpea rhizobia cultured in media of pH 4.5, whereas little was produced at pH 7.0, or by an acid-sensitive strain. In studies under- taken with R. tropici we have shown only limited short-term cytoplasmic pH regulation, although cytoplasmic pH in cells grown at pH 5.0 rose from pH 5.48 to 6.60 over a 5-h period, and was paralleled by changes in the pH of the external medium. Cells subject to acidity maintained higher levels of potassium (Aarons and Graham 1991) and glutamate (Graham et al. 1992), a response similar to that found in osmotically stressed cells (see p. 478).

Studies on the genetic basis of tolerance to low pH sug- gest a chromosomal rather than a plasmid location for pH genes (Chen et al. 1991; Graham et al. 1992). Goss et al. (1990) cloned DNA from the acid-tolerant R. meliloti WSM419 into pMMB33, and transformed this cosmid into Escherichia coli. When clones were probed with DNA car- rying Tn5 and flanking sequences from Tn5-containing pH-sensitive mutants of R. meliloti WSM419, one clone car- ried DNA complementing for intracellular pH maintenance. In similar studies we have used clone banks of the acid- tolerant R. tropici strain LTMR1899 in pSLTP205 (Cevallos et al. 1989) and pVK102 (Vargas et al. 1990) to transform the acid-sensitive bean strain CE3, and have identified at least two clones capable of complementing this strain to growth at pH 4.5 (P.H. Graham, unpublished data). "Acid- shock" proteins have also been shown in both TA1 (Glenn

et al. 1986) and LTMR1899 (Aarons and Graham 1991), and the demonstration that pH-complemented clones produce one or more shock proteins different from those found in the acid-sensitive recipient could give early information on the gene product. A practical approach to the development of more effective acid-tolerant strains is that of Chen et al. (1991), who first cured an acid-tolerant clover strain of its sym plasmid, then transferred to it the sym plasmid of a superior nitrogen-fixing strain.

The failure of legumes to nodulate under acid-soil condi- tions is common (Brockwell 1962; Morales et al. 1973; Rice et al. 1977), especially in soils of pH less than 5.0. The inability of some rhizobia to persist under such conditions is one cause of nodulation failure (Graham et al. 1982; Rice et al. 1977; Lowendorf and Alexander 1983), but poor nodulation can occur even where a viable Rhizobium population can be demonstrated. Evans et al. (1980) found the nodulation of Pisum sativum 10 times more susceptible to acidity than either rhizobial multiplication or plant growth. Despite this, elevated inoculation levels have enhanced nodulation response in some studies (Munns 1968; Pijnenborg et al. 1991). The pH-sensitive stage in nodula- tion occurs early in the infection process, nodulation only being inhibited when plants are exposed to acidity during the period of root-hair extension and curling (Munns 1968; Lie 1969). Rhizobium attachment to root hairs is one of the stages affected (Vargas and Graham 1988; Caetano-Anolles et al. 1989), but Richardson et al. (1988b) have also shown that nod gene induction by 7,4'-dihydroxyflavone in Rhizobium leguminosarum bv. trifolii is inhibited by low pH. The same authors (Richardson et al. 1988a) obtained exudates from cultivars of subterranean clover grown in media of pH 3.0-8.0 and showed that ability to induce nod gene activity in clover rhizobia was maximal in samples grown at pH 5.0-6.0, and was significantly reduced at pH 4.5.

Few studies have compared the survival in acid soil of strains differing in pH tolerance. However, acid tolerance has usually (Graham et al. 1982; Rice 1982; Hartel and Alexander 1983; Howieson and Ewing 1986), but not always (Bromfield and Jones 1980; Howieson et al. 1988; Howieson and Ewing 1989), been associated with enhanced nodula- tion in acid soil. Thus Keyser et al. (1979) successfully iden- tified about 65% of the cowpea strains that were sym- biotically acid sensitive through their inability to grow at pH 4.5, and Graham et al. (1982) obtained a strong response to inoculation with an acid-tolerant strain under conditions where an equally effective, but acid-sensitive strain gave no response. These and similar reports (Lindstrom and Myllyniemi 1987; Wood and Shepherd 1987; Taylor et al. 1991) must balance those that suggest that a negative cor- relation exists between acid-pH tolerance and strain effec- tiveness (Van Schreven 1972; Richardson and Simpson 1989).

Differences in pH tolerance do influence strain com- petitiveness. Thus nodule occupancy of the bean strains Car37 and Car43 was reduced from 22 and 65%, respec- tively, in a soil of pH 5.1, to only 3 and 5% after the soil was limed to pH 6.7 (Voss et al. 1984), while nodule occupancy by Car04 increased from only 12% at pH 5.1 to 60% at pH 6.2. Similar results have been reported in other studies (Dughri and Bottomley 1983; Ramos and Boddey 1987; Vargas and Graham 1988).

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Host cultivar - strain interactions at acid pH deserve fur- ther study. Munns et al. (1979) noted that nodulation and nitrogen fixation by some strains of Bradyrhizobium at acid pH differed with the cultivar of mung bean used, while Vargas and Graham (1988) identified several acid-tolerant bean cultivars and, in studies with both acid-tolerant and sensitive rhizobia, showed that only one of the symbionts needed to be acid tolerant for good nodulation at pH 4.5. Use of the acid-tolerant R. meliloti WSM419 with Medicago polymorpha has extended the area of acid soils in Western Australia that can be sown to annual legumes by some 350 000 ha since 1985 (Howieson et al. 1988). We believe that signaling between host and Rhizobium is a factor in these interactions and are currently using lac2 fusions to explore cultivar and strain response.

Aluminum and manganese toxicities Availability of the highly toxic ~ 1 ~ ' ion in soil or solu-

tion is markedly affected by pH, with a sharp decline in availability between pH 4.5 and 4.8. When tested at an appropriate pH, rhizobia can vary markedly in tolerance to aluminum. For Bradyrhizobium, 25 of 38 strains that grew at pH 4.5 were also resistant to 50 pM A1 (Keyser and Munns 1979b), while 33 of 78 bean strains tolerated pH 4.5 plus 100 pM A1 (Vargas and Graham 1988). Some but not all Al-tolerant strains also tolerated 200 pM Mn, while some strains tolerated 200 pM Mn but not 100 pM Al. Some clover rhizobia (Whelan and Alexander 1986) and fast- growing Lotus rhizobia also tolerate 50 pM A1 (Wood et al. 1988), but other clover strains tested have proved sensitive (Wood and Cooper 1988). Johnson and Wood (1990) reported that A1 was taken up and bound to the DNA of both sensitive and tolerant strains, but that DNA synthesis by the tolerant R. loti was not affected. By contrast Richardson et al. (1 988b) found even 7.5 pM A1 to depress nod gene expression at pH 4.8.

Plant species vary markedly in tolerance to aluminum and manganese, with some significantly more affected by these ions than are the rhizobia. Reduction in the growth of Medicago sativa was found at only 8- 18.5 pM Al, but other N-fertilized tropical legumes showed no growth inhibition at 74 pM A1 (Munns 1965; Andrew et al. 1973). Nodula- tion appears more sensitive to A1 than is plant growth, Car- valho et al. (1981, 1982) and Murphy et al. (1984) each reporting inhibition of nodulation at 25 pM Al. At pH 4.5 and with 0.5 mM Ca, nodulation in cowpea was delayed 4 days by concentrations of 12.7 pM Al, and nodule num- ber and dry weight were severely depressed (Alva et al. 1 990).

Calcium and phosphorus requirements Reported Ca requirements for unstressed cells of Rhizobium

and Bradyrhizobium are quite low; Norris (1958, 1959) found no evidence of a need for calcium; Vincent (1962) and Keyser and Munns (1979b) noted calcium requirements of only 25-50 pM. Calcium appears significantly more impor- tant in cells exposed to low pH. O'Hara et al. (1989) found 1-2 mM Ca needed for cytoplasmic pH maintenance in acid- sensitive strains of R. meliloti, while Beck and Munns (1984, 1985) noted that P-limited cells or cells grown at low pH needed Ca for P mobilization in the cell. Calcium also plays a role in cell wall integrity. Ca-depleted cells of R. legumin- osarum are swollen, lack rigidity, and express an additional somatic antigen normally blocked by side chains of the

lipopolysaccharide 0 antigen (Vincent and Humphrey 1968; De Maagd et al. 1989). Monoclonal antibodies have also been reported that recognize a lipopolysaccharide antigen whose detection is regulated by pH (Kannenberg and Brewin 1989; Wood et al. 1989). If the calcium-depletion and pH-engendered antigens are one and the same, a role for calcium in the maintenance of cell wall integrity at low pH could also be argued. We have some evidence of change in lipopolysaccharide structure under acid conditions, and are looking to relate this to calcium availability.

Suboptimal calcium concentrations both delay the appear- ance of nodules and limit nodulation. For guar and pigeon pea the critical calcium level for nodule formation in flowing-solution culture is more than 50 pM, whereas peanut and cowpea were well nodulated at 2 pM (Bell et al. 1989). In studies with the specific calcium chelator ethylene glycol- bis(P-aminoethyl ether)N, N, N' , N' -tetraacetic acid (EGTA), addition of agar blocks containing only 6 pM EGTA to the initially susceptible root region of the plant reduced nodula- tion from 87 to 32070, but only when the EGTA was applied during the first day after inoculation (Pijnenborg et al. 1990). Calcium requirements for nodulation are increased by low pH. With alfalfa plants grown at pH 6.0 Munns (1 970) achieved abundant nodulation with 0.1 -0.2 mM Ca, whereas when the pH was dropped to pH 5.2, 6.4 mM Ca was needed to achieve comparable results. With cowpea, Alva et al. (1990) found no effect of pH (4.5-5.5) and calcium concentration (0.05-2.5 mM) on plant growth, but nodulation and nodule development were strongly depressed when both pH and Ca were low. Calcium-dependent cell surface components affect attachment of Rhizobium to root- hair cells (Smit et al. 1987, 1989; Caetano-Anolles et al. 1989), while addition of 10 mM calcium increased nod gene induction activity by white and subterranean clover 5- to 10-fold at pH 4.5 (Richardson et al. 1988a). Calcium also enhanced nod gene expression at pH 4.8-5.2 (Richardson et al. 1988b).

Phosphate in the soil solution varies from lo- ' to l o p 7 M, and in the rhizosphere of plants or in old tropical soils can fall to M (Wild 1988). While Rhizobium and Bradyrhizobium strains differ marked1 in P utilization effi- J ciency, most require at least 5 x 10- M phosphorus, and some may require more than 1 pM phosphorus (Cassman et al. 1981 b; Beck and Munns 1985). Thus rhizobial P defi- ciency in soil and rhizosphere is a real possibility, especially under acid conditions where solution P may be precipitated in the presence of aluminum.

Slow-growing strains again appear more low P tolerant than are fast growers, with R. meliloti, in particular, P requiring (Beck and Munns 1984, 1985). Rhizobia grown at luxury levels of P can accumulate this substance to levels as high as 2.4% (Cassman et al. 198 la; Beck and Munns 1984), using these reserves on transfer to P-limited media. Rhizobium meliloti strains may be an exception to this, requiring high calcium levels for P mobilization (Beck and Munns 1985).

When E. coli is subject to phosphorus deprivation in cul- ture, genes coding for functions associated with phosphate scavenging are derepressed and function to promote P uptake. This topic is extensively reviewed by Torriani-Gorini et al. (1987), but low P responses include modification of the outer membrane pore proteins to specifically enhance phosphate diffusion through the outer membrane; binding

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of phosphate by the periplasmic pore protein; and transport of phosphate through the cytoplasmic membrane via the phosphate specific transport (pst) system. To date, parallel studies with the rhizobia are lacking, although Smart et al. (1984b) have shown that phosphate-limited cultures of both fast- and slow-growing rhizobia do take up phosphate 10- to 180-fold faster than cells grown with adequate P, and that inducible alkaline phosphatase activity was detectable in P-limited cells of Rhizobium, but not Bradyrhizobium (Smart et al. 1984a, 1984b). P-repressible and P-inducible bands also occur in the periplasmic protein profiles of rhizobial cells grown with different levels of P (Smart et al. 1984~).

The pho regulon is but one example of how micro- organisms respond to stress. Stock et al. (1989) describe a number of two-component systems by which bacteria detect and respond to external stimuli. Each involves a protein kinase, commonly a membrane receptor protein that uses ATP to phosphorylate itself at a histidine residue, and a response regulator that in turn accepts the phosphoryl group. In the case of phosphate limitation, the major protein kinase is PhoR, while the PhoB regulator controls expression of alkaline phosphatase activity, the production of phosphate- specific porins, and the pst system (Stock et al. 1989). To date the only comparable work with Rhizobium has been with regulation of nitrogenase activity with nitrogen supply (Sundaresan et al. 1983; Buikema et al. 1985). Additional studies on both P utilization and adaptive response are needed.

Leguminous species in flowing-solution culture can dif- fer in their P requirement for growth from 0.8 to 3 pM (Fist et al. 1987). Differences between cultivars have also been reported (Schettini et al. 1987; Burauel et al. 1990) but are difficult to sustain under field conditions, where differences in level of infection with mycorrhizal fungi can be a complicating factor. Studies on the molecular basis of response to P deprivation in plants are beginning, Goldstein et al. (1989) noting the production of at least six proteins following the transfer of suspension-cultured tomato cells to low-P medium.

Phosphorus appears essential for both nodulation and nitrogen fixation. Significant delays have been reported in the infection of primary soybean roots by P-limited cells of Bradyrhizobium japonicum (Mullen et al. 1988), although in some host-strain combinations P stress subsequently resulted in enhanced nodule mass and shoot N. Leung and Bottomley (1987) also found that P deprivation delayed nodulation in subterranean clover, with this delay overcome ei.ther by P supplementation or by raising the medium pH to 6.5. Nodules are strong sinks for phosphorus and range in P content from 0.72 to 1.12% (Hart 1989a, 1989b). This is a higher level than found in roots, shoots, or even young mature leaves at all but luxury levels of P. As a consequence, fixation-dependent plants will require more of this element than those supplied combined nitrogen (Cassman et al. 198 1c; Israel 1987) and may appear P inefficient (Israel and Rufty 1988). Nodulation, nitrogen fixation, and specific nodule activity can all be directly related to P supply (Graham and Rosas 1979; Jakobsen 1985; Singleton et al. 1985).

Osmotic stress and salinity Rhizobium and Bradyrhizobium strains show marked

variation in salt tolerance. A number are inhibited by 100 mM salt (Singleton et al. 1982; Yelton et al. 1983; Zhang et al. 1991) but growth at salt concentrations of more than 300 mM has been reported with strains of R. meliloti (Graham and Parker 1964; Sauvage et al. 1983), R. fredii (Yelton et al. 1983), and R. tropici (P.H. Graham and M.L. Ferrey, unpublished data), and some alfalfa, Acacia, Prosopis, and Leucaena strains will tolerate 500 mM NaCl (Sauvage et al. 1983; Zhang et al. 1991). For a chickpea Rhizobium, similar results were obtained when potassium, calcium, or magnesium salts were used, although the chloride of each was more toxic than the sulfate (Elsheikh and Wood 1989). It is interesting that, as with pH, the principal sources of salt tolerance reside in little-studied and under-collected organisms poorly represented in most cul- ture collections. It certainly makes a case for the continued collection of the rhizobia and the characterization of new isolates.

For rhizobia from Prosopis and Medicago, exposure to salt or osmotic stress results in production of intracellular glutamate (Hua et al. 1982; Botsford 1984; Botsford and Lewis 1990), with levels in Rhizobium WRlOOl reaching 288 pM per gram dry weight of cells, and accounting for 88% of the intracellular amino acid pool (Hua et al. 1982). Enhanced glutamate accumulation has also been reported for recombinants of USDA110 containing DNA from a fast- growing and salt-tolerant strain of R. fredii (Yang and Li 1989). While such increases in glutamate level are striking, Botsford and Lewis (1990) make the point that they represent only 10% of the capacity of the cell for glutamate produc- tion, and would necessitate only small changes in the spe- cific activity of one or more of the enzymes involved in glut amate synthesis. Gonzalez-Gonzalez et al. (1 990) showed very limited effects of salt on the enzymes involved in glutamate synthesis but did find that potassium accumula- tion inhibited GOGAT and GDH activity, while enhancing that of glutamine synthetase. Potassium also accumulates under osmotic stress in R. meliloti (Le Rudulier and Bernard 1986; Botsford and Lewis 1990), R. fredii (Yelton et al. 1983), and the rhizobia from Leucaena leucocephala (Yap and Lim 1983), so it is possible that as in E. coli (Booth and Higgins 1990; Welsh et al. 1991) the accumulation of potassium glutamate serves as a signal for secondary responses. One such response already identified in the rhizobia is the intracellular accumulation of glycine betaine (Sauvage et al. 1983; Bernard et al. 1986; Le Rudulier and Bernard 1986; Tombras-Smith et al. 1988). Le Rudulier and Bernard (1986) found that addition of 1 mM glycine betaine significantly reduced the doubling time of strains of R. meliloti following their exposure to NaCl, but did not affect that of the other more sensitive Rhizobium strains tested. On exposure to NaCl, salt-tolerant strains increased their rate of uptake of glycine betaine and were able to syn- thesize this substance more rapidly from choline, but showed decreased catabolism of glycine betaine (Bernard et al. 1986; Le Rudulier and Bernard 1986; Tombras-Smith et al. 1988). Trehalose may also play a role in osmoregulation (Elsheikh and Wood 1990), while the dipeptide N-acetylglutaminyl- glutamine amide may also accumulate in Rhizobium as the result of osmotic stress (Tombras-Smith and Smith 1989). Osmotic shock proteins such as found in E. coli (Barron et al. 1986; Botsford 1990) have yet to be reported in Rhizobium or Bradyrhizobium. In E. coli, Botsford (1990)

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found 41 proteins whose production was increased at least 10-fold in salt-stressed cells, and 6 of these each accounted for more than 1 % of the radioactive label.

Hypoosmotic adaptation has also been reported in Rhizobium (Dylan et al. 1990), Agrobacterium (Miller et al. 1986; Cangelosi et al. 1989, 1990), and Bradyrhizobium (Tully et al. 1990). For R. meliloti, growth in low osmolarity medium is accompanied by the synthesis of periplasmic cyclic 6-(1-2)glucans, while in Bradyrhizobium the oligo- saccharides synthesized are smaller and are differently linked (Miller et al. 1990; Tully et al. 1990). The possible role of such glucans in nodule development (Dylan et al. 1986) has created considerable interest.

Legumes and the process of nodule initiation are both more highly sensitive to salt or osmotic stress than are the rhizobia (Russell 1976; Tu 198 1 ; Zahran and Sprent 1986; Velageleti et al. 1990). Soybean root hairs showed little curl- ing or deformation when inoculated with B. japonicum in the presence of 170 mM NaCl, and nodulation was com- pletely suppressed by 210 mM NaCl (Tu 198 1). Similarly Zahran and Sprent (1986) found bacterial colonization and root-hair curling of Vicia faba reduced in the presence of 50- 100 mM NaCl or 100-200 mM polyethylene glycol (PEG), with the proportion of root hairs containing infec- tion threads reduced 30 and 52% under NaCl and PEG, respectively. Effects of salt stress on nitrogen fixation have been examined in several studies (Lauter et al. 1981; Rai and Prasad 1983; Velalgaleti et al. 1990) but, in each case, have imposed treatments at planting, compounding nodulation and nitrogen fixation effects. Rai and Prasad identified two nitrosoguanidine mutants of the salt-sensitive strain RL5 which showed significantly enhanced nodulation and nitrogen fixation under salt stress; while Velalgaleti et al. (1990) found that 5 of 16 soybean varieties they tested were somewhat tolerant of 80 mM salt. In grafts between salt- "tolerant" and sensitive cultivars, the root stock determined degree of nodulation and nitrogen fixation.

Temperature Temperature can affect rhizobial persistence in inoculants

during shipment or in storage, can influence survival in soil, and can limit both nodulation and nitrogen fixation. Results will vary with the strain used, the severity of the stress and the period for which it is imposed, and moisture conditions. Temperature studies undertaken have not always been rele- vant to the field situation.

For most rhizobia the temperature optimum for growth in culture is from 28 to 31°C, with many unable to grow below 10 or at 37°C. However, arctic rhizobia are known to grow well at 10°C (Ek-Jander and Fahraeus 1971; Caudry-Reznick et al. 1986), while the optimum for R. meliloti is 35°C (Allen and Allen 1950) and 90% of cowpea strains obtained from the hot, dry environment of the sahel savannah grew well at 40°C (Eaglesham and Ayanaba 1984). Strain adaptation of high temperature has also been reported by Wilkins (1967), Hartel and Alexander (1984), and Karanja and Wood (1988b). The latter authors found a high percentage of the strains that persisted at 45°C to have lost their infectiveness, and attributed this to plasmid curing. High soil temperatures could contribute to the fre- quency of noninfective isolates in soil, Segovia et al. (1991) noting that such noninfective isolates actually outnumbered those that were infective in the rhizosphere of bean. Heat-

shock proteins have been shown in Rhizobium (Aarons and Graham 1991) but have not been studied in detail.

Rhizobial survival in soil exposed to high temperatures has been shown to vary with the montmorillonite content of the soil (Marshall 1964), to be greater in soil aggregates than in nonaggregate soil (Ozawa et al. 1988), and to be favored by dry rather than moist conditions. For unsterilized peat inoculants for clover, Date and Roughley (1 977) report weekly log death rates in stored peat from 0.04 at 5°C to 0.094 at 25"C, although survival of Centrosema and Desmodium rhizobia in peat was better at 26 than at 4°C (Roughley 1982). Ten inoculant strains examined by Somasegaran et al. (1984) showed a gradual decline in population during 8 weeks incubation at 37"C, while expo- sure to 46°C was lethal to all strains in less than 2 weeks. Decreases in the infectivity of cowpea rhizobia following storage at 35°C have also been shown (Wilson and Trang 1980). In tests of inoculants shipped to tropical and sub- tropical locations, maximum temperatures experienced were from 26 to 45"C, with peat populations only reduced below desirable levels in 5 of 53 shipments (Somasegaran et al. 1984).

Temperature affects root-hair infection, bacteroid dif- ferentiation and nodule structure, and the functioning of the legume root nodule (Roughley and Dart 1969; Roughley 1970; Roughley and Dart 1970). Gibson (1 967) found 7°C the minimum temperature for nodulation of subterranean clover, with time to initial nodulation increased as tempera- tures declined below 22°C. Similarly, Hardarson and Gareth Jones (1979) noted restriction of strain 75str on S184 white clover markedly affected by temperature, while the pea cultivar Iran was resistant to nodulation by R. legumin- osarum at 20 but not 26°C (Lie 1971). Cultivar variation in temperature response is also evident in the results of Rennie and Kemp (1981), who showed that the bean variety Kentwood failed to nodulate at 10°C and took 32 days to initial nodulation at 12"C, while cv. Aurora nodulated in 23 and 21 days at 10 and 12"C, respectively. Strain dif- ferences in nodulating ability at low temperature have also been shown between arctic- and temperate-zone rhizobia (Ek-Jander and Fahraeus 1971), while Rice and Olsen (1988) found dual strain occupancy reduced from 63% at 8°C to 2% at 25"C, with the proportion of nodules occupied by strain NRG185 alone increased from 9 to 75Vo over the same temperature range.

High soil temperatures will also delay nodulation or restrict it to the subsurface region, where temperatures are not as extreme. Munns et al. (1977) found that alfalfa plants grown in desert environments in California maintained few nodules in the top 5 cm of soil but were extensively nodulated below this depth, while Graham and Rosas (1978) observed fewer nodules close to the surface in spaced plant- i n g ~ than in plantings with dense canopies. Nodulation of soybean was markedly inhibited at 42°C (12-h day) and 45°C (9-h day) (La Favre and Eaglesham 1986), with no correla- tion between a strain's ability to grow at high temperature and to induce nodulation under temperature stress. This finding is in contrast to the earlier results of Munevar and Wollum (198 l a 1981 b) but may reflect differences in the temperatures used. Heavier than normal inoculation rates may be required under such high temperature conditions. In Puerto Rico taproot and total nodulation in soybean was enhanced at 10-fold inoculation rates (Smith and del Rio

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Escurra 1982), while Smith et al. (1981) found no nodules formed when the inoculant was applied at less than cells per centimetre of row, and nodule number increased with application rate to cells per centimetre of row.

As temperatures increase toward an optimum, time to initiation of nitrogen fixation will decrease and initial rates of fixation increase. Thus Graham (1979) found early fixa- tion in Phaseolus vulgaris greater at 35:25 OC (1ight:dark) than at 25: 15OC, with plants at the lower temperature suf- fering nitrogen deficiency to 28 days after planting. How- ever, the nitrogen fixation of plants at the higher tempera- ture peaked and declined earlier, with much greater overall fixation at 25: 15 than at 35:25OC. Most high-temperature studies have either not used diurnal fluctuations in temper- ature or have been carried out at a single point in the grow- ing season. Use of pots rather than of more difficult to heat soil beds may also have confounded results. Piha and Munns (1987) noted that bean nodules developed at low tempera- tures were unaffected by increases in daily temperature to 35OC, although those formed at 35OC were small and had low specific activity, but Hernandez-Armenta et al. (1989) found that transferring nodulated bean plants from 26 to 35°C daily temperature markedly inhibited nitrogen fixa- tion. Soybean plants appear somewhat more heat tolerant, nitrogen fixation only being severely inhibited by daytime temperatures greater than 4.1 OC, and enhanced at 36OC (La Favre and Eaglesham 1987).

Summary Until perhaps 10 years ago, studies of environmental stress

and the legume - Rhizobium or Bradyrhizobium symbiosis were restricted to defining the problem, and to using physi- cal or chemical amendments to overcome it, i.e., mulching to reduce soil temperature or liming to ameliorate soil acidity. The identification of bacterial strains and in some cases host cultivars that are tolerant to these stresses opens the way for alternate, lower cost solutions to these prob- lems. Molecular studies of host-strain interaction and studies of stress sensing and response are being initiated, and it is likely that the next decade will be a period of intense activ- ity in this field. By the year 2000 we should have identified and assigned functions to the genes associated with pH and osmotic stress tolerance, as well as having a good under- standing of the basis for host-strain interaction under stress. Although we will not be able to eliminate many of the stresses currently limiting crop production under low-input condi- tions, we should be able to identify better host-strain com- binations and develop other ways to minimize the impact of stress, giving rise to a more sustainable agriculture.

Acknowledgements The support of the USAID Beadcowpea Collaborative

Research Support Program and the Minnesota Soybean Research and Promotion Council is gratefully acknowledged.

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