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Nodulation and N2 fixation by soybean inoculated with salt-tolerant rhizobia or salt-sensitive bradyrhizobia in saline soil
Pergamon
0038-0717(!34)00147-2
Soil Bid. Biochem. Vol.27, No. 4/5, pp. 657-661, 1995 Copyright 0 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0038-0717/95 $9.50 + 0.00
NODULATION AND N2 FIXATION BY SOYBEAN INOCULATED WITH SALT-TOLERANT RHIZOBIA OR SALT-SENSITIVE BRADYRHIZOBIA IN SALINE SOIL E. A. E. ELSHEIKH* Department
and M. WOOD7
of Soil Science, The University of Reading, London Road, Reading RGI SAQ, England
Summary-Nodulation of soybean by a salt-tolerant RI&o&urn strain USDA 208 and a salt-sensitive Bradyrhizobium strain RCR 3407 (CBl809) was evaluated in saline soil in the glasshouse. No significant differences in shoot and root dry weight, nodule number and nodule weight between the salt-tolerant Rhizobiumstrain and the salt-sensitive Bradyrhizobiumstrain were found. Nodulation of soybean was reduced more than 50% by the equivalent of 34.2 mM NaCl in soil. The specific activity of nodules formed by both strains was also reduced by salinity. Nodulation was more sensitive than plant growth to salinity. The salt-sensitive strain was more effective than the salt-tolerant strain under non-saline conditions, but the salt-tolerant strain fixed more N, than the salt-sensitive strain under saline conditions.
INTRODUCTION
Wood (1990), together with details of growth media and experimental procedures for screening strains for tolerance of salinity.
Salinity has been reported to reduce shoot and root weights, nodulation and total plant N in legumes such as chickpea (Lauter et al., 1981), soybean
(Singleton and Bohlool, 1984) and faba bean (Yousef and Sprent, 1983; Zahran and Sprent, 1986). Nodule initiation in the legume-I&izobium (Bradyrhizobium) symbiosis involves a complex interaction between the host root, rhizobial strain and environment. The process of nodule initiation in soybean was reported to be extremely sensitive to NaCl. A reduction in nodulation of 50% compared to maximum nodule number and nodule weight occurred with 26.6 mM NaCl in solution culture (Singleton and Bohlool, 1984). However, Tu (1981) found that up to 102 mM NaCl in solution culture did not result in a decline in nodule number for soybean. Although there are reports on salt-tolerance in rhizobia and bradyrhizobia no comparisons have been made of nodulation and Nz fixation between salt-tolerant and salt-sensitive strains under saline conditions. The aims of our experiment using saline soil in the glasshouse were to determine the effect of salts on: (i) nodulation of a salt-tolerant cultivar of soybean using salt-tolerant rhizobia and salt-sensitive bradyrhizobia; and (ii) plant N uptake and biological Nz fixation. MATERIALS
AND METHODS
Strains of Rhizobium and Bradyrhizobium and screening for salt -tolerance Details of strains of rhizobia and bradyrhizobia for soybeans (Glycine max) are described in Elsheikh and *Present address: Department of Biochemistry and Soil Science, University of Khartoum, Khartoum, Sudan. tAuthor for correspondence.
Evaluation of salt -tolerant and salt -sensitive strains under saline conditions @ot experiment)
One kilogramme amounts of soil (O-20 cm Rowland series collected from the University of Reading Farm at Sonning) were placed in 13 cm dia pots and planted with one seedling of soybean cultivar TGX 814-49E (obtained from the International Institute for Tropical Agriculture, Ibadan, Nigeria, and identified in preliminary experiments as relatively salt-tolerant). Four salinity levels using a mixture of NaCl and CaCl, (0.25 Ca:Na on molar basis) were used to give 0, 5.0 and 10.0 dS m-’ (equivalent to 0, 51.3 and 195.9mM NaCl, respectively). Seedlings were inoculated immediately after transplanting withRhizobium strain USDA 208 or Bradyrhizobium strain RCR 3407 (CB1809) (lo’-lo6 cfu g-’ soil). The salt was added in the irrigation water (every 3 days) which started 3-5 days after transplanting. There was an uninoculated control. Irrigation with saline water continued for 3 weeks and thereafter the plants received only tap water until the end of the experiment. At the beginning of the experiment, to each pot a solution of CaNO, (5 at. % “N) was added to give the equivalent of 20 kg N ha-‘. Four replicates were used per treatment, and pots were kept in a glasshouse at 22 + 3°C with 14-15 h daylight. The plants were harvested 4 or 6 weeks after starting the salt treatment. Dry weights of shoots (leaves and stems) and roots were measured, as well as nodule number and average nodule weight per plant. The harvested samples were dried at 8o”C, ground to a fine powder and analysd for N and lSN content using a Carlo Erba NA1500 Nz analyser linked to a VG Isogas 602E mass spectrometer. The
657
658
E. A. E. Elsheikh and M. Wood
proportion of soybean N derived from the atmosphere (pNdfa) was estimated using the following equation (Fried and Middleboe. 1977):
Table 2. The effect of salinity (N&I + C&I,) on shoot and root dry weight (g plant ‘) of soybean cultivar TGX 814-498 either uninoculated. inoculated with salt-tolerant strain USDA 208 or inoculated with salt-sensitive strain RCR 3407 (CBlSG9) harvested at 6 weeks after starting the salt treatment
pNdfa = I - (“N at. % excess inoc!
Salt level (dS m -‘) lnoculatlon
15Nat. % excess uninoc). where inoc is inoculated soybeans and uninoc is uninoculated soybeans (which were not nodulated). An estimate of the amount of N1 fixed was calculated, based on weighted mean enrichments of the shoots plus roots. using the following equation: Nz fixed = legume total N x pNdfa.
AND DISCL’SSION
Selection of’salt -tolerant struins ,fi)r so?$7ean Screening strains of rhizobia and bradyrhizobia fol multiplication in liquid-defined medium containing different concentrations of NaCl has indicated that R. ,fiedii are generally more tolerant of salinity than B. japonicum (Elsheikh and Wood. 1990). Table I shows data for two strains of rhizobia and two strains of bradyrhizobia. USDA 208 was selected as an example of a salt-tolerant strain, and RCR 3407 (CB1809) as an example of a salt-sensitive strain. Evaluation qf’salt -tolerunt under saline conditions
and suit -sensitive strains
Soybean plants survived and grew at all salinity levels. Salinity significantly reduced the dry weights of both shoots and roots (P < 0.001) in both pot experiments (Table 2). Salinity has been reported to reduce shoot and root weights in soybean (Singleton and Bohlool, 1984; G&tan and Mass, 1988). Inoculation significantly increased shoot (P < 0.05) and root (P < 0.001) dry weights of soybean plants (Table 2). There was a significant inter-
Table I. The response of IWO >tran\ of Klxohirm~ and tuo stra,ns of Bra~lvh~x~hiu~~~to different concentrations of call in defined liquid medium NaCl (M) stram
0
0.14
0.21
0.34
R. firrlii USDA 20X USDA 217
/ +
i c
1 +
+
B. ,‘1Jxvw U,?, RCR 3X24 RCR 3407
+ +
i
5.0
10.0
3.57 3.85 3.73
3.27 3.30 3.33
(b) Roots (SE 5 0.08 9) I .53 I.10 1.74 I .50 2.17 I.12
0.84 I.18 I .02
(a)
shoots(SE& 0.3 g)
Uninoculated USDA 208 RCR 3407 Uninoculated USDA 208 RCR 3407
4.63 5.48 6.26
Four replicates per treatment.
In addition the amount of N: fixed was estimated by the N difference method (Lyon and Bizzell, 1934). in which the total N yield of the uninoculated soybean was subtracted from that of the inoculated soybean and the difference assumed to be derived from N, fixation.
RESULTS
0
0.51
+. growth: -. no growth (wIthIn 14 days itt 25 C). Two observations per value. Data from Elshelkh and Wood (1990).
action between salt and inoculation for both shoots (P < 0.05) and roots (P < 0.05). This interaction resulted from the increase in dry matter of shoot and roots in plants inoculated with the salt-sensitive strain in non-saline conditions compared to that of the salt-tolerant and uninoculated treatments. No nodules were found on roots of soybean plants in the uninoculated control treatment. Salinity significantly (P < 0.001) reduced the total nodule number per plant (Fig. I). The effect was less severe after 6 weeks compared to 4 weeks. No significant differences were observed in nodulation between the salttolerant and the salt-sensitive strains. There were no differences between the strains at all levels of salinity. Hafeez et a/. (I 988) reported that nodulation of Vigna radiuta was reduced by about half, by 5.0dS mm’ when compared to 1.4 dS rn--‘. However, they also found that nodulation was completely depressed when salinity was increased to 10.0 dS mm’, regardless of the plant growth stage, unlike the data reported here for soybean. The processes of nodule initiation in soybean is reported to be extremely sensitive to NaCI. A reduction in nodule number and weight of 50% occurred with 26.6 mM NaCl in the rooting medium (Singleton and Bohlool, 1984). In our study, nodulation of soybeans was reduced by more than 50% at 34.2 mM NaCl in solution culture (data not shown) and in soil (Fig. I). Tu (1981) reported that the failure of soybean to nodulate at high salinity might be attributed to shrinkage of root hairs. Salinity also interferes with nodule initiation in chickpea (Balasubramanian and Sinha, 1976a; Lauter et al., 1981). mung bean (Balasubramanian and Sinha, l976b) and field bean (Yousef and Sprent, 1983). Inoculation significantly increased the total N yield of both shoots (P < 0.001) and roots (P < 0.05), as shown in Table 3. However, salinity significantly decreased the N yield of both shoots (P < 0.001) and roots (P c 0.01). There was a significant interaction between salinity and the strain used for total N in both shoots (P < 0.05) and roots (P < 0.05). The salt-sensitive (Brudyrhizobium) strain yielded more N in shoots and roots than the tolerant (Rho&urn)
Salinity
strain under non-saline conditions, but there was no difference under saline conditions (Table 3). The non-nodulated uninoculated plants were used as the non-fixing reference plant for estimating the amount of N, fixation. The proportion of N derived from the atmosphere (pNdfa) in the shoots was greater than for roots of inoculated soybean plants (Table 4). However, the values for pNdfa were significantly decreased by 10.0 dS mm’ (105.9 mM NaCl) for
Nodule number
SE=?
plants inoculated with either the salt-tolerant or the salt-sensitive strain (Table 4). This confirms that N, fixation was more sensitive to salinity than plant growth. This was partly due to a reduction in number of nodules formed (Fig. 1) and also to a reduction in N, fixed per nodule. The specific activity of nodules formed by USDA 208 after 6 weeks was 1.3 and 0.4 mg N nodule-’ for 0 and 10 dS m-l, respectively; and the value for RCR 3407 (CB1809) was 1.9 and
4.43 .s
*pcm
USDA 208
659
effect on N, fixation
SpX*,
RCR 3407
(salt-tolerant1
(salt-sensitive)
7a 60 M 40 3a 20 10 0
salinity (tis m-l) 4 weeks 6 weeks
Nodule weight
s~=t45.5
g spcnr
rpC*tr
USDA 208
salinity (ds In-’ ) 4 weeks 6 weeks
(salt-tolerant)
RCR 3407
0
salinity 4 weeks
(ds
m-’) 6 weeks
5
(salt-sensitive)
10
0
5
10
salinity (ds m-l )
4 weeks
6 weeks
Fig. 1. The effect of salinity (CaCI, + NaCl) on nodulation of soybean variety TGX 8 14-49E inoculated with either the salbtolerant Rhizobkm strain USDA 208 or with salt-sensitive Bradyrhizobium strain RCR 3407(CB1809)at different times after starting the salt treatment. Three replications per treatment.
660
E. A. E. Elsheikh and M. Wood
Table 3. The effect of salinity (NaCI + CaCI,) on total N (mg plant -‘) for shoots and roots of soybean cultivar TGX 814-49E either uninoculated, inoculated with salt-tolerant Rhimbium strain USDA 208 or salt-sensitive Bradyrhtobium strain RCR 3407 (CBl809) harvested at 6 weeks after starting the salt treatment Salt level (dS m-l) Inoculation
0.0
5.0
USDA 208 RCR 3407
34.75 53.94 49.08
Uninoculated USDA 208 RCR 3407
(b) Roots (SE + 7.8 mg N plant-‘) 18.90 17.10 38.90 34.70 59.80 27.50
14.50 20.10 16.30
Four replicates per treatment
0.2 mg N nodule--’ for 0 and 10 dS m ‘, respectively. These differences were not associated with a decrease in the size of the nodules (data not shown). Similar results have been observed for soybean (Wilson, 1970). However, Yousef and Sprent (1983) reported different results for field bean in which 100 mM NaCl increased specific nodule activity. The results reported here indicate that R. fkdii strain USDA 208 and B. japonicum strain RCR 3407 (CB 1809) were able to form effective symbiosis with the soybean cultivar TGX 814-49E under non-saline conditions. Although fast-growing soybean rhizobia (R. fredii) form nodules on most soybean varieties, their symbiotic effectiveness may be different from the typical slow-growing soybean bradyrhizobia (B. japonicum) (Keyser et al., 1982). N, fixation results for whole plants obtained by the N difference method were similar to results from the isotope dilution method (Table 5). Data from both methods
Table 4. The effect of sahnity (N&I +&Cl,) on lSN (at. % excess) content, the proportion of N derived from fixation by inoculated plants (pNdfa) and the amount of N, fixed by soybean cultivar TGX 814-498 either uninoculated (U), inoculated with salt-tolerant Rhimbium strain USDA 208 (T) or salt-sensitive Bradyrhkobium strain RCR 3407 (S) harvested 6 weeks after starting the salt treatment Inoculation
Salt level (dSm ‘)
‘SN
pNdfa content
N, fixation (mg N plant ‘j
U T S U T S U T S
(a) Shoots (SE = fO.20 for pNdfa) 0 2.110 NA 0 0.800 0.618 0 0.650 0.693 5 2.350 NA 5 I .070 0.540 5 1. I70 0.502 IO 2.260 NA IO I .670 0.253 IO I.930 0.147
NA 66.50 87.60 NA 40.00 34.40 NA 14.00 7.20
U T S U T S U T s
(b) Roots (SE = kO.19 for pNdfa) 0 1.600 NA 0 0.770 0.520 0 0.560 0.648 5 1.800 NA 5 0.710 0.582 5 0.890 0.508 1.800 NA IO IO I.1 IO 0.384 IO I.250 0.304
NA 20.20 38.80 NA 20.20 14.00 NA 7.70 5.00
Four replicates per treatment. NA = not applicable.
Salt treatment (dS m .‘)
10.0
(a) Shoots (SE * 7.9 mg N plant-‘) 47.47 46.88 107.66 73.81 126.44 68.42
Uninoculated
Table 5. Comparison of values for N, fixed (mg plant ‘) obtained using N difference (ND) or “N isotope dilution (ID) method for soybean cultivar TGX 814-49E inoculated with either salt-tolerant Rhizobiumstrain USDA 208 (T) or salt-sensitive Brudyrhizobium strain RCR 3407 (S) and subject to different salinity (N&I + C&I,) concentrations for 6 weeks
Method
Strain
0
5
IO
ID ID
T S
86.7 126.4
60.2 48.4
21.7 12.2
ND ND
T S
80.0 118.9
44.5 31.9
24.8 16.2
indicated that there was an interaction between salinity and the strain used. This is because the saltsensitive strain fixed more N, than the salt-tolerant strain under non-saline conditions, whereas the salttolerant strain fixed more Nz than the salt-sensitive strain under saline conditions. In contrast to these data, Yelton et al. (1983) found no differences between R. fredii strain USDA 191 (salt-tolerant) and B. japonicum strain USDA 110 (salt-sensitive) in terms of the total nodule number per plant, nodule weight and total plant weight for soybean under non-saline conditions. However, the salt-sensitive strain fixed more N, than the salttolerant strain under non-saline conditions. The cultivar used may also have an important effect on the performance of these salt-tolerant strains (Raovelagaleti and Marsh, 1989). Overall, these data indicate that although salinity reduces nodulation and N, fixation in soybean, the use of a salt-tolerant strain under saline conditions can produce an increase in biological N, fixation when compared to a salt-sensitive strain. AcknoM,ledgemenrs-awe thank the Sudanese Government for financial support. We are also grateful to Dr H. Keyser (USDA) for supplying cultures of soybean rhizobia.
REFERENCES
Balasubramanian V. and Sinha S. K. (1976a) Nodulation and nitrogen fixation in chickpea (Cicer ariehum L.) under salt stress. Journal q/ Agricultural Science, Cambridge 87, 465466. Balasubramanian V. and Sinha S. K. (I 976b) Effects of salt stress on growth, nodulation and nitrogen fixation in cowpea and mung beans. Physiologia Plantarum 36, 197-200. Elsheikh E. A. E. and Wood M. (1990) Salt effects on survival and multiplication of chickpea and soybean rhizobia. Soil Biology & Biochemistry 22, 343-347. Fried M. and Middelboe V. (1977) Measurement of amount of nitrogen fixed by a legume crop. Plant and Soil 47, 713-71s. Grattan S. R. and Mass E. V. (1988) Effect of salinity on phosphate accumulation and injury in soybean: I. Influence of CaCIJNaCI ratios. Plant and Soil 105,25-32. Hafeez F. Y.. Aslam Z. and Malik K. A. (1988) Effect of salinity and inoculation on growth, nitrogen fixation and nutrient uptake of Vigna radiara (L.) Wilczek. P/ant and Soil 106, 3-8. Keyser H. H., Bohlool B. B.. Hu T. S. and Weber D. F.
661
Salinity effect on N, fixation (1982) Fast-growing rhizobia isolated from root nodules of soybean. Science 215, 1631-1632. Lauter D. J., Munns D. N. and Clarkin K. L. (1981) Salt response of chickpeas influenced by N supply. Agronomy Journal 13, 961-966.
Research 21, 571-582.
Lyon T. L. and Bizzell T. A. (1934) A comparison of several legumes with respect to N accretion. Journal of the American Society
qf Agronomy
Wilson J. R. (1970) Response to salinity in glycine: VI. Some effects of a range of short term salt-stresses on the growth, nodulation and nitrogen fixation of Glycine whightii (Formerly javanica). Australian Journal of Agricultural
26, 651-656.
Raovelagaleti R. and Marsh S: (1989) Influence of host cultivars and Bradyrhizobium strains on the growth and symbiotic performance of soybean under salt-stress. Plant and Soil 119, 133-138. Singleton P. W. and Bohlool 8. B. (1984) Effect of salinitv on the nodule formation by soybean. Piant Physiology Ii, 72-76.
Tu J. C. (1981) Effect of salinity on Rhizobium-root hair interaction, nodulation and growth of soybean. Canadian Journal of Plant Science 61, 231-239.
Yelton M. M., Yang S. S., Edie S. A. and Lim S. T. (1983) Characterization of an effective salt tolerant fast growing strain of Rhizobium japonicum. Journal of General Microbiology 129, 1537-1547. Yousef A. N. and Sprent J. I. (1983) Effects of NaCl on growth, nitrogen incorporation and chemical composition of inoculated and NH,NO, fertilized Vicia faba IL.) slants. Journal of Botanv, 34., _ Experimental . -941-930.’
-
Zahran H. H. and Sprent J. I. (1986) Effects of sodium chloride and polyethylene glycol on root-hair and nodulation of Vicia faba L. plants by Rhizobium leguminosarum. Planta 167, 303-309.
0038-0717(!34)00147-2
Soil Bid. Biochem. Vol.27, No. 4/5, pp. 657-661, 1995 Copyright 0 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0038-0717/95 $9.50 + 0.00
NODULATION AND N2 FIXATION BY SOYBEAN INOCULATED WITH SALT-TOLERANT RHIZOBIA OR SALT-SENSITIVE BRADYRHIZOBIA IN SALINE SOIL E. A. E. ELSHEIKH* Department
and M. WOOD7
of Soil Science, The University of Reading, London Road, Reading RGI SAQ, England
Summary-Nodulation of soybean by a salt-tolerant RI&o&urn strain USDA 208 and a salt-sensitive Bradyrhizobium strain RCR 3407 (CBl809) was evaluated in saline soil in the glasshouse. No significant differences in shoot and root dry weight, nodule number and nodule weight between the salt-tolerant Rhizobiumstrain and the salt-sensitive Bradyrhizobiumstrain were found. Nodulation of soybean was reduced more than 50% by the equivalent of 34.2 mM NaCl in soil. The specific activity of nodules formed by both strains was also reduced by salinity. Nodulation was more sensitive than plant growth to salinity. The salt-sensitive strain was more effective than the salt-tolerant strain under non-saline conditions, but the salt-tolerant strain fixed more N, than the salt-sensitive strain under saline conditions.
INTRODUCTION
Wood (1990), together with details of growth media and experimental procedures for screening strains for tolerance of salinity.
Salinity has been reported to reduce shoot and root weights, nodulation and total plant N in legumes such as chickpea (Lauter et al., 1981), soybean
(Singleton and Bohlool, 1984) and faba bean (Yousef and Sprent, 1983; Zahran and Sprent, 1986). Nodule initiation in the legume-I&izobium (Bradyrhizobium) symbiosis involves a complex interaction between the host root, rhizobial strain and environment. The process of nodule initiation in soybean was reported to be extremely sensitive to NaCl. A reduction in nodulation of 50% compared to maximum nodule number and nodule weight occurred with 26.6 mM NaCl in solution culture (Singleton and Bohlool, 1984). However, Tu (1981) found that up to 102 mM NaCl in solution culture did not result in a decline in nodule number for soybean. Although there are reports on salt-tolerance in rhizobia and bradyrhizobia no comparisons have been made of nodulation and Nz fixation between salt-tolerant and salt-sensitive strains under saline conditions. The aims of our experiment using saline soil in the glasshouse were to determine the effect of salts on: (i) nodulation of a salt-tolerant cultivar of soybean using salt-tolerant rhizobia and salt-sensitive bradyrhizobia; and (ii) plant N uptake and biological Nz fixation. MATERIALS
AND METHODS
Strains of Rhizobium and Bradyrhizobium and screening for salt -tolerance Details of strains of rhizobia and bradyrhizobia for soybeans (Glycine max) are described in Elsheikh and *Present address: Department of Biochemistry and Soil Science, University of Khartoum, Khartoum, Sudan. tAuthor for correspondence.
Evaluation of salt -tolerant and salt -sensitive strains under saline conditions @ot experiment)
One kilogramme amounts of soil (O-20 cm Rowland series collected from the University of Reading Farm at Sonning) were placed in 13 cm dia pots and planted with one seedling of soybean cultivar TGX 814-49E (obtained from the International Institute for Tropical Agriculture, Ibadan, Nigeria, and identified in preliminary experiments as relatively salt-tolerant). Four salinity levels using a mixture of NaCl and CaCl, (0.25 Ca:Na on molar basis) were used to give 0, 5.0 and 10.0 dS m-’ (equivalent to 0, 51.3 and 195.9mM NaCl, respectively). Seedlings were inoculated immediately after transplanting withRhizobium strain USDA 208 or Bradyrhizobium strain RCR 3407 (CB1809) (lo’-lo6 cfu g-’ soil). The salt was added in the irrigation water (every 3 days) which started 3-5 days after transplanting. There was an uninoculated control. Irrigation with saline water continued for 3 weeks and thereafter the plants received only tap water until the end of the experiment. At the beginning of the experiment, to each pot a solution of CaNO, (5 at. % “N) was added to give the equivalent of 20 kg N ha-‘. Four replicates were used per treatment, and pots were kept in a glasshouse at 22 + 3°C with 14-15 h daylight. The plants were harvested 4 or 6 weeks after starting the salt treatment. Dry weights of shoots (leaves and stems) and roots were measured, as well as nodule number and average nodule weight per plant. The harvested samples were dried at 8o”C, ground to a fine powder and analysd for N and lSN content using a Carlo Erba NA1500 Nz analyser linked to a VG Isogas 602E mass spectrometer. The
657
658
E. A. E. Elsheikh and M. Wood
proportion of soybean N derived from the atmosphere (pNdfa) was estimated using the following equation (Fried and Middleboe. 1977):
Table 2. The effect of salinity (N&I + C&I,) on shoot and root dry weight (g plant ‘) of soybean cultivar TGX 814-498 either uninoculated. inoculated with salt-tolerant strain USDA 208 or inoculated with salt-sensitive strain RCR 3407 (CBlSG9) harvested at 6 weeks after starting the salt treatment
pNdfa = I - (“N at. % excess inoc!
Salt level (dS m -‘) lnoculatlon
15Nat. % excess uninoc). where inoc is inoculated soybeans and uninoc is uninoculated soybeans (which were not nodulated). An estimate of the amount of N1 fixed was calculated, based on weighted mean enrichments of the shoots plus roots. using the following equation: Nz fixed = legume total N x pNdfa.
AND DISCL’SSION
Selection of’salt -tolerant struins ,fi)r so?$7ean Screening strains of rhizobia and bradyrhizobia fol multiplication in liquid-defined medium containing different concentrations of NaCl has indicated that R. ,fiedii are generally more tolerant of salinity than B. japonicum (Elsheikh and Wood. 1990). Table I shows data for two strains of rhizobia and two strains of bradyrhizobia. USDA 208 was selected as an example of a salt-tolerant strain, and RCR 3407 (CB1809) as an example of a salt-sensitive strain. Evaluation qf’salt -tolerunt under saline conditions
and suit -sensitive strains
Soybean plants survived and grew at all salinity levels. Salinity significantly reduced the dry weights of both shoots and roots (P < 0.001) in both pot experiments (Table 2). Salinity has been reported to reduce shoot and root weights in soybean (Singleton and Bohlool, 1984; G&tan and Mass, 1988). Inoculation significantly increased shoot (P < 0.05) and root (P < 0.001) dry weights of soybean plants (Table 2). There was a significant inter-
Table I. The response of IWO >tran\ of Klxohirm~ and tuo stra,ns of Bra~lvh~x~hiu~~~to different concentrations of call in defined liquid medium NaCl (M) stram
0
0.14
0.21
0.34
R. firrlii USDA 20X USDA 217
/ +
i c
1 +
+
B. ,‘1Jxvw U,?, RCR 3X24 RCR 3407
+ +
i
5.0
10.0
3.57 3.85 3.73
3.27 3.30 3.33
(b) Roots (SE 5 0.08 9) I .53 I.10 1.74 I .50 2.17 I.12
0.84 I.18 I .02
(a)
shoots(SE& 0.3 g)
Uninoculated USDA 208 RCR 3407 Uninoculated USDA 208 RCR 3407
4.63 5.48 6.26
Four replicates per treatment.
In addition the amount of N: fixed was estimated by the N difference method (Lyon and Bizzell, 1934). in which the total N yield of the uninoculated soybean was subtracted from that of the inoculated soybean and the difference assumed to be derived from N, fixation.
RESULTS
0
0.51
+. growth: -. no growth (wIthIn 14 days itt 25 C). Two observations per value. Data from Elshelkh and Wood (1990).
action between salt and inoculation for both shoots (P < 0.05) and roots (P < 0.05). This interaction resulted from the increase in dry matter of shoot and roots in plants inoculated with the salt-sensitive strain in non-saline conditions compared to that of the salt-tolerant and uninoculated treatments. No nodules were found on roots of soybean plants in the uninoculated control treatment. Salinity significantly (P < 0.001) reduced the total nodule number per plant (Fig. I). The effect was less severe after 6 weeks compared to 4 weeks. No significant differences were observed in nodulation between the salttolerant and the salt-sensitive strains. There were no differences between the strains at all levels of salinity. Hafeez et a/. (I 988) reported that nodulation of Vigna radiuta was reduced by about half, by 5.0dS mm’ when compared to 1.4 dS rn--‘. However, they also found that nodulation was completely depressed when salinity was increased to 10.0 dS mm’, regardless of the plant growth stage, unlike the data reported here for soybean. The processes of nodule initiation in soybean is reported to be extremely sensitive to NaCI. A reduction in nodule number and weight of 50% occurred with 26.6 mM NaCl in the rooting medium (Singleton and Bohlool, 1984). In our study, nodulation of soybeans was reduced by more than 50% at 34.2 mM NaCl in solution culture (data not shown) and in soil (Fig. I). Tu (1981) reported that the failure of soybean to nodulate at high salinity might be attributed to shrinkage of root hairs. Salinity also interferes with nodule initiation in chickpea (Balasubramanian and Sinha, 1976a; Lauter et al., 1981). mung bean (Balasubramanian and Sinha, l976b) and field bean (Yousef and Sprent, 1983). Inoculation significantly increased the total N yield of both shoots (P < 0.001) and roots (P < 0.05), as shown in Table 3. However, salinity significantly decreased the N yield of both shoots (P < 0.001) and roots (P c 0.01). There was a significant interaction between salinity and the strain used for total N in both shoots (P < 0.05) and roots (P < 0.05). The salt-sensitive (Brudyrhizobium) strain yielded more N in shoots and roots than the tolerant (Rho&urn)
Salinity
strain under non-saline conditions, but there was no difference under saline conditions (Table 3). The non-nodulated uninoculated plants were used as the non-fixing reference plant for estimating the amount of N, fixation. The proportion of N derived from the atmosphere (pNdfa) in the shoots was greater than for roots of inoculated soybean plants (Table 4). However, the values for pNdfa were significantly decreased by 10.0 dS mm’ (105.9 mM NaCl) for
Nodule number
SE=?
plants inoculated with either the salt-tolerant or the salt-sensitive strain (Table 4). This confirms that N, fixation was more sensitive to salinity than plant growth. This was partly due to a reduction in number of nodules formed (Fig. 1) and also to a reduction in N, fixed per nodule. The specific activity of nodules formed by USDA 208 after 6 weeks was 1.3 and 0.4 mg N nodule-’ for 0 and 10 dS m-l, respectively; and the value for RCR 3407 (CB1809) was 1.9 and
4.43 .s
*pcm
USDA 208
659
effect on N, fixation
SpX*,
RCR 3407
(salt-tolerant1
(salt-sensitive)
7a 60 M 40 3a 20 10 0
salinity (tis m-l) 4 weeks 6 weeks
Nodule weight
s~=t45.5
g spcnr
rpC*tr
USDA 208
salinity (ds In-’ ) 4 weeks 6 weeks
(salt-tolerant)
RCR 3407
0
salinity 4 weeks
(ds
m-’) 6 weeks
5
(salt-sensitive)
10
0
5
10
salinity (ds m-l )
4 weeks
6 weeks
Fig. 1. The effect of salinity (CaCI, + NaCl) on nodulation of soybean variety TGX 8 14-49E inoculated with either the salbtolerant Rhizobkm strain USDA 208 or with salt-sensitive Bradyrhizobium strain RCR 3407(CB1809)at different times after starting the salt treatment. Three replications per treatment.
660
E. A. E. Elsheikh and M. Wood
Table 3. The effect of salinity (NaCI + CaCI,) on total N (mg plant -‘) for shoots and roots of soybean cultivar TGX 814-49E either uninoculated, inoculated with salt-tolerant Rhimbium strain USDA 208 or salt-sensitive Bradyrhtobium strain RCR 3407 (CBl809) harvested at 6 weeks after starting the salt treatment Salt level (dS m-l) Inoculation
0.0
5.0
USDA 208 RCR 3407
34.75 53.94 49.08
Uninoculated USDA 208 RCR 3407
(b) Roots (SE + 7.8 mg N plant-‘) 18.90 17.10 38.90 34.70 59.80 27.50
14.50 20.10 16.30
Four replicates per treatment
0.2 mg N nodule--’ for 0 and 10 dS m ‘, respectively. These differences were not associated with a decrease in the size of the nodules (data not shown). Similar results have been observed for soybean (Wilson, 1970). However, Yousef and Sprent (1983) reported different results for field bean in which 100 mM NaCl increased specific nodule activity. The results reported here indicate that R. fkdii strain USDA 208 and B. japonicum strain RCR 3407 (CB 1809) were able to form effective symbiosis with the soybean cultivar TGX 814-49E under non-saline conditions. Although fast-growing soybean rhizobia (R. fredii) form nodules on most soybean varieties, their symbiotic effectiveness may be different from the typical slow-growing soybean bradyrhizobia (B. japonicum) (Keyser et al., 1982). N, fixation results for whole plants obtained by the N difference method were similar to results from the isotope dilution method (Table 5). Data from both methods
Table 4. The effect of sahnity (N&I +&Cl,) on lSN (at. % excess) content, the proportion of N derived from fixation by inoculated plants (pNdfa) and the amount of N, fixed by soybean cultivar TGX 814-498 either uninoculated (U), inoculated with salt-tolerant Rhimbium strain USDA 208 (T) or salt-sensitive Bradyrhkobium strain RCR 3407 (S) harvested 6 weeks after starting the salt treatment Inoculation
Salt level (dSm ‘)
‘SN
pNdfa content
N, fixation (mg N plant ‘j
U T S U T S U T S
(a) Shoots (SE = fO.20 for pNdfa) 0 2.110 NA 0 0.800 0.618 0 0.650 0.693 5 2.350 NA 5 I .070 0.540 5 1. I70 0.502 IO 2.260 NA IO I .670 0.253 IO I.930 0.147
NA 66.50 87.60 NA 40.00 34.40 NA 14.00 7.20
U T S U T S U T s
(b) Roots (SE = kO.19 for pNdfa) 0 1.600 NA 0 0.770 0.520 0 0.560 0.648 5 1.800 NA 5 0.710 0.582 5 0.890 0.508 1.800 NA IO IO I.1 IO 0.384 IO I.250 0.304
NA 20.20 38.80 NA 20.20 14.00 NA 7.70 5.00
Four replicates per treatment. NA = not applicable.
Salt treatment (dS m .‘)
10.0
(a) Shoots (SE * 7.9 mg N plant-‘) 47.47 46.88 107.66 73.81 126.44 68.42
Uninoculated
Table 5. Comparison of values for N, fixed (mg plant ‘) obtained using N difference (ND) or “N isotope dilution (ID) method for soybean cultivar TGX 814-49E inoculated with either salt-tolerant Rhizobiumstrain USDA 208 (T) or salt-sensitive Brudyrhizobium strain RCR 3407 (S) and subject to different salinity (N&I + C&I,) concentrations for 6 weeks
Method
Strain
0
5
IO
ID ID
T S
86.7 126.4
60.2 48.4
21.7 12.2
ND ND
T S
80.0 118.9
44.5 31.9
24.8 16.2
indicated that there was an interaction between salinity and the strain used. This is because the saltsensitive strain fixed more N, than the salt-tolerant strain under non-saline conditions, whereas the salttolerant strain fixed more Nz than the salt-sensitive strain under saline conditions. In contrast to these data, Yelton et al. (1983) found no differences between R. fredii strain USDA 191 (salt-tolerant) and B. japonicum strain USDA 110 (salt-sensitive) in terms of the total nodule number per plant, nodule weight and total plant weight for soybean under non-saline conditions. However, the salt-sensitive strain fixed more N, than the salttolerant strain under non-saline conditions. The cultivar used may also have an important effect on the performance of these salt-tolerant strains (Raovelagaleti and Marsh, 1989). Overall, these data indicate that although salinity reduces nodulation and N, fixation in soybean, the use of a salt-tolerant strain under saline conditions can produce an increase in biological N, fixation when compared to a salt-sensitive strain. AcknoM,ledgemenrs-awe thank the Sudanese Government for financial support. We are also grateful to Dr H. Keyser (USDA) for supplying cultures of soybean rhizobia.
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