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Role of siderophores in chickpea (Cicer arietinum L.) — Rhizobium symbiosis
Microbiol. Res. (\998) 153,47-53
©
Gustav Fischer Verlag
Role of siderophores in chickpea (Cicer arietinum L.) - Rhizobium symbiosis Monika Dhul, Sunita Suneja, K. R. Dadarwal Department of Microbiology, CCS Haryana Agricultural University, Hisar-125 004, India Accepted: January 18, 1998
Abstract Strains of Rhizobium ciceri (wild type as well as mutants) were screened for their ability to produce siderophores under cultural conditions. All strains except a mutant Ca401RW5 were found to be siderophore producers. A standard strain Ca 181 which is being used for large scale production as inoculant for chickpea, found to be a siderophore producer, was used for standardization of conditions for siderophore production. Only hydroxamate type of siderophores were detected under cultural conditions. The level of hydroxamate production increased linearly during log phase of growth up to 8 days. Addition of iron in the medium resulted in decrease in hydroxamate production and at 500 J.lM iron level, the hydroxamate level decreased by 40%. In other strains tested, decrease in hydroxamate production varied from 30 to 75% whereas in one strain Ca85AZ3 this level of iron completely inhibited hydroxamate production. Strain Ca 181 was most effective in terms of symbiotic nitrogen fixation in absence as well as added iron. Addition of iron stimulated nodule fresh weight in almost all the strains tested. It also resulted in increased symbiotic effectivity in terms of nitrogen gain per plant although the response varied with the bacterial strain. Key words: Rhizobium ciceri - siderophore - hydroxamate iron
Introduction Iron is essential for growth of all living organisms. Although, it is fourth most abundantly available element on the earth's crust but its availability to plants as well as microorganisms is hindered due to its easy chemical oxidation. It forms insoluble ferric salts, mainly hydroxides under acidic conditions and phosphates under neuCorresponding author: K. R. Dadarwal
tral to alkaline conditions. To solubilize and sequester iron, most of the microorganisms produce extracellular low molecular weight iron transport agents called siderophores (Neilands 1981) which are either phenolates or hydroxamates. Siderophore production in Rhizobium assumes special interest as iron is required not only for nodule formation but also for synthesis of key proteins required in nitrogen fixation viz. nitrogenase, hydrogen uptake hydrogenase and leghaemoglobin. How iron is obtained by the infecting bacterium inside the host system is not clear but it has been suggested that differences in nodule development under iron deficient conditions may be due to varying abilities of different strains of Rhizobium to acquire iron for nodule initiation and development (O'Hara eta!' 1988). Hydroxamate type siderophores are stable in soil over a wide range of pH and promote iron uptake by plants (Powell et a!. 1982; Reid et al. 1984). Scanty informations are available regarding production ofhydroxamate siderophores by rhizobia (Carson et al. 1992, 1994; Suneja eta!' 1992, 1994). Present study was carried out to investigate prevalence and nature of siderophores in strains of Rhizobium ciceri, the root nodule bacteria of chickpea (Cicer arietinum L.) to understand the role of siderophores in symbiosis. This micro symbiont is highly host specific.
Materials and methods Rhizobium strains. Rhizobium ciceri strains and some mutants of standard strains used in the present studies were procured from the Department of Microbiology, Chaudhary Charan Singh Haryana Agricultural UniverMicrobio!. Res. 153 (1998) 1
47
sity, Hisar. These strains were maintained by periodic transfer on Yeast extract mannitol agar slants (Vincent 1970). Screening of Rhizobium ciceri strains for siderophore production. The universal chemical assay as described by Schwyn and Neilands (1987) was used for qualitative detection of siderophores in different strains using chrome azurol S dye (CAS) agar plates for bacterial growth. A loopful each of 7d old cultures from Yema slopes was spotted on the medium plates and incubated at 30°C for 5 -7 d. Observations were taken for colony dia and halo zone formed around the colony to calculate halo zone area as described by Van-Rossum et al. (1994). Optimization of conditions. Optimization of conditions for maximum siderophore production with respect to culture media, stage of growth and iron concentration in the medium was done with Rhizobium ciceri strain Ca181. The strain was pregrown for 7 don YEMA slants and the cells from slants were suspended in sterilized saline. The cell suspension (0.1 ml) was used to inoculate 20 ml YEM broth (Vincent 1970) or Complete medium broth (Modi et al. 1985) in 50 ml flasks. The flasks were incubated at 300C on a rotary shaker for 4 d. The broth culture was then used to inoculate in fresh broth in multiple replications and incubated at 30°C on a rotary shaker. At different time intervals, three flasks of each medium were used for estimating protein and siderophores (hydroxamate and catechol type). Protein was estimated by the method of Lowry et al. (1951). The method of Csaky (1948) modified by Suneja and Lakshminarayana (1993) was used for estimation of hydroxamate type of siderophores and the method of Arnow (1937) was used for the estimation of catechol type of siderophores. Iron concentration. Optimum iron concentration was determined by growing the culture for 8 d in Complete medium (Modi et al. 1985) with different levels (0-500 11M) of iron in the form of FeCI3 . Siderophore production by different Rhizobium ciceri strains. The strains were grown in 20 ml of Complete medium broth without iron and with iron (500 11M FeCI3) in 50 ml conical flasks in triplicate and incubated at 30°C for 8 d on a rotary shaker. Protein and siderophores were estimated with appropriate methods described earlier. Total siderophores in culture supernatants after centrifugation were also estimated using chrome azurol S (CAS) assay method as described by Van-Rossum et al. (1994). Effect of iron on symbiotic effectivity. Healthy seeds of chickpea var C235 were surface sterilized with acidic alcohol (cone. sulphuric acid: ethanol, 7: 3, v/v) followed 48
Microbiol. Res. 153 (1998) 1
by thorough washing with sterilized water (Dadarwal et al. 1985). The surface sterilized seeds were inoculated with iron starved broth culture of ten selected Rhizobium isolates and their mutants. The inoculated seeds were sown in autoclaved chillum jar assemblies (Dahiya and Khurana 1981) containing washed river sand and Sloger's nitrogen free mineral salt solution (Sloger 1969) without iron and with iron. Uninoculated seeds were sown as controls. The jars were kept in a net house under day light conditions. The Sloger's nitrogen free mineral salt solution was modified to have 0 (control), 50 and 166 11M of FeCI 3• Thus for each strain two levels of iron with control (no iron) were kept to see the effect of added iron on symbiotic effectivity. The relative symbiotic effectivity of different strains was determined at 60 d of plant growth by taking into consideration the number of nodulus formed per plant, fresh weight of nodules, plant dry weight, nitrogenase activity and total plant nitrogen. Nitrogenase activity in nodules was determined by measuring acetylene reduction activity (Hardy et al. 1968). The nodules were detached from the roots after measuring acetylene reduction activity, counted and then weighed for fresh nodule weight. Total nitrogen content of the plants was determined calorimetrically using Nessler's reagent (Linder 1944).
Results Screening of Rhizobium ciceri strains for siderophore production Table 1 shows relative siderophore production by different strains of Rhizobium cieeri on CAS agar plates. All strains except a mutant Ca401RW5 showed production of siderophore. Strain CaI81-17-3B failed to grow on the CAS agar plates. Siderophore production was "corrected" for growth by dividing the halo zone area by the colony area (RIG; Table 1). Nine strains showed ratio between 1.7 to 3.2 which were considered to be low producers, 8 showed a ratio of 3.6 to 5.0 (moderate siderophore producers whereas 3 strains namely Ca534St8, Ca339 and Ca85St7 showed ratio more than 7.0 and were considered to be high siderophore producers. Effect of culture media and stage of growth on siderophore production Production of hydroxamate and catechol type of siderophore was studied in Rhizobium ciceri strain Ca181 to see the effect of medium composition and stage of bacterial growth. Catechol type of siderophore was absent at all stages of growth in both Complete and YEM medium. Maximum hydroxamate production was observed
Table 1. Relative siderophore production by different strains of Rhizobium ciceri on CAS agar plates Wild strain! mutant
Siderophore production Colony growth (G) (dia)
Halo zone (H) (dia)
Growth to halo zone ratio (G: H)
1.8 1.5 1.5 1.0 2.5 1.0 1.0 1.0 1.0 1.5 1.5 1.5 1.5 2.0 3.0 2.0 1.5 1.0 No growth 2.0 0.5 2.0
3.5 3.0 3.0 2.0 8.0 1.5 1.5 1.5 7.0 2.5 2.5 4.0 2.0 4.0 4.5 4.5 3.0 1.8
3.78 4.00 4.00 4.00 10.24i 2.25 2.25 2.25 49.00i 2.77 2.77 7.1 Ji 1.77 4.00 2.25 5.06 4.00 3.24
~
~
Table 2. Production of hydroxamate siderophore in Rhizobium ciceri strains as affected by iron* Wild Strain/ Mutant
Protein (/lg ml- I )
Hydroxamate (/lg Nmg- I protein)
FeFe FeFe-
93 146
6.56 2.95
222 267
1.39 0.93
CA 181 St4
FeFe+
145 170
8.62 2.82
CA 181 M 115
FeFe FeFe+
77 98
3.60 0.81
90 105
3.50 2.28
Ca 85 St 7
FeFe+
110 175
2.50 1.13
Ca339
FeFe
107 219
2.20 0.57
Ca410R
FeFe FeFe+
123 235
1.21 0.53
235 256
0.72
Ca401 RW 5
FeFe+
248 272
(-) (-) (-)
Ca534 St8
FeFe
205 230
7.81 5.34
G =Growth expressed as colony diameter (mm) H =Siderophore production expressed as halo diameter (mm) H/G =Halo area (0.5 H)2 divided by growth area (0.5 G)2 ~ =Not detected i =High value caused by small colony size
Ca32
FeFe
195 219
5.98 4.56
Ca 19 ANal
FeFe FeFe+
190 210
5.71 4.11
179 195
4.57 2.50
at the end of log phase on 8th d with corresponding production level of 5.7 and 1.8 flg N mg- I protein in Complete and YEM medium respectively (Fig. 1).
Ca534
FeFe+
172 185
3.65 2.40
Ca2 St9
FeFe
137 150
3.35 2.02
Effect of iron
CV4
FeFe+
130 141
2.53 1.88
Ca2
FeFe FeFe+
120 136
2.00 1.45
99 112
1.87 1.25
FeFe FeFe FeFe
87 105
(-) (-)
110 175
3.41 1.37
123 235
1.21 0.51
Ca 13 Ca32 Ca46 Ca 171 Ca339 CV4 Ca85 CA 85 Az 3 CA85 St7 Ca 85 St 10 Ca534 Ca 534 St 8 Ca2 Ca2 St 9 Ca181 Ca 181 M 115 Ca 181 St4 Ca 181-7-3B Ca 181-17-3B Ca40IR Ca40l RW5 Ca 19ANal
4.5
5.06
~
~
3.0
2.25
Fig. 2 shows effect of iron on hydroxamate siderophore production in strain Ca 181. Production of hydroxamate decreased with increase in concentration of iron in the medium. There was 40% decrease in production of hydroxamate at 500 flM iron concentration. Shah et ai. (1993) observed complete inhibition of hydroxamate siderophore in Azospirillum iipoferum at 5 flM concentration of iron.
Ca 181 CA 181-17-3 B
Ca85
Ca 85 Az 3
Ca13
Ca 181-17-3 B Ca46 Ca 85 St 10
Hydroxamate production in different Rhizobium ciceri strains
Ca171
Table 2 shows production of hydroxamate siderophores in Rhizobium ciceri strains. All strains except Ca401RW5 and Ca46 showed production of hydroxamate siderophores. In the presence of iron (500 flM), hy-
* Iron added in form of FeC1 3 (500 /lM) in Complete medium. Results are average of three replicates.
Microbial. Res. 153 (1998) 1
49
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Fig. 1. Effect of medium composition on bacterial cell protein and hydroxamate siderophore in relation to time of incubation. 0 Protein in Complete medium, [J [J Hydroxamate in Complete medium, 8 8 Hydroxamate yield in Complete medium, 0 ---- 0 Protein in YEM medium, [J [J Hydroxamate in YEM medium, 8 --- 8.Hydroxamate yield in YEM medium.
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Fig. 2. Effect of iron concentration on hydroxamate type of siderophore production in Rhizobium ciceri strain Ca 181 at 8 d of growth. -[J- Protein, -8- Hydroxamate yield. 50
Microbiol. Res. 153 (1998) 1
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Fig. 3. Siderophore production by Rhizobium ciceri strains in Complete medium with or without added Fe. and assayed with CAS supernatant assay (With shuttle). Table 3. Effect of inoculation of siderophore producers and non-producers strains of Rhizobium ciceri on symbiotic effectivity in chickpea cv. C 235 at 60 d under chillum jar conditions Inoculant Strain
Nodule No. (pi-I)
Nodule fresh weight Shoot dry weight (mg pi- I) (mg pi- I)
ARA (n moles h- I pi- I)
Total plant nitrogen (mg pi-I)
Fe- Fe 1 Fe2
Fe-
Fe-
Fe-
Fe 1 Fe2
Control Ca 181
19
20
2
11
4
28
27
Cal 181-17-3 B 5
10
9
Ca 181 M 115 Ca 181 St4
322
Fe-
Fe 1
Fe2
229
265
319
503
483
361
371
478
12
158
267
272
298
103
296
257
283
371
368
15.64
9
29
137
109
279
319
295
5.95
17
112
114
136
275
288
323
11.05
36.04
Ca85
7
Ca 85 Az 3
3
13
14
34
164
186
244
266
388
53.40
Ca 85 St 7
7
12
13
112
148
164
251
287
327
8.87
Ca339
2
3
3
29
32
35
265
353
389
15 .51
Ca401 R
5
8
11
85
123
226
261
278
289
7.66
Ca401 RW 5
2
18
24
20
189
263
263
324
427
9
Fe 1
Fe2
4.1 (1.8) 114.84 179.20 8.2 (2.4) 7.15 10.46 5.8 (2.2) 27.20 45 .33 6.2 (2.2) 7.05 37 .05 5.3 (1.9) 20.07 43 .71 6.6 (2.4) 55.63 55.63 4.8 (2.0) 48.50 50.20 5.7 (2.3) 28.60 33.11 5.5 (2.1) 49.67 66.83 5.7 (2.2) 21 .53 59.71 5.5 (2.1)
Fe 1
Fe2
5.6 (2.1) 9.2 (2.5) 7.6 (2.8) 8.9 (2.4) 7.0 (2.2) 7.2 (2.5) 7.1 (2.7) 7.1 (2.5) 8.4 (2.4) 6.9 (2.5) 8.1 (2.5)
7.0 (2.2) 11.9 (2.5) 8.3 (2.9) 9.5 (2.6) 8.2 (2 .8) 8.7 (2.7) 10.8 (2.8) 8.1 (2.5) 9.7 (2.5) 7.2 (2.5) 10.6 (2.5)
Fe- - Iron absent; Fe 1 - 50 flM iron; Fe 2 - 166 flM iron. Figures in parentheses indicate % N. Values are average of three replicates. Microbiol. Res. 153 (1998) I
51
droxamate level decreased approximately from 30 to 75% in different strains. In strain Ca85Az3 this level of iron completely inhibited hydroxamate production. Above strains were further tested for total siderophore production using CAS supernatant assay with shuttle. All strains except Ca401RW5 were found to be siderophore positive (Fig. 3). The responses of many tested strains were relatively weak i.e. small b.-A 630 values. Symbiotic effectivity of Rhizobium ciceri strains under chillum jar conditions
Table 3 shows effect of inoculation of siderophore producers and nonproducer strains on symbiotic effectivity in chickpea cv 235. Nodulation, plant dry weight and total nitrogen per plant were more in treatments in which iron was used than control without iron thus clearly showing the role of iron in symbiotic nitrogen fixation. Maximum plant dry weight, ARA per plant and total nitrogen per plant was observed with inoculation of strain Ca181. This strain also produced maximum nodule fresh weight indicating clearly that higher effectivity of this strain was due to higher nodule biomass production. Other strains which responded to iron with regard to gain in total plant nitrogen were Ca181St4, Ca181M115, Ca181-17-3B and Ca85Az3. Strain Ca181St4 produced maximum amount of hydroxamate siderophore and strain Cal81M115 produced maximum amount of total siderophores. In both the strains the relative nitrogen fixation potential taking into consideration the relative nodule biomass was also higher which indicate involvement of siderophore production in symbiotic nitrogen fixation.
Discussion Detection of siderophores using chrome azurol S (CAS) agar plate is based on the ability of siderophore to act as chelator with varying affinity for iron. The presence of this iron chelator is indicated by decolorization of blue coloured ferric CAS complex, resulting in an orange halo around the colonies on CAS plates. In the present study, this approach was followed and 22 strains and mutants of Rhizobium ciceri were screened for siderophore production, out of which 20 were found to be positive, one was negative and one showed no growth on CAS agar plate. YEM medium and Complete medium were used for detection of hydroxamate or catechol type of siderophores in Rhizobium ciceri strain Ca181. Catechol type of siderophore was absent in this strain but the strain produced hydroxamate type of siderophore which was maximum at the end of log phase. Absence of catechol type of siderophores has been reported earlier also in dif52
Microbial. Res. 153 (1998) 1
ferent species of Rhizobium (Carson et al. 1992; Suneja et al. 1992). These workers found production of hydro xamate type of siderophores in the rhizobia studied. Similar to our observations production of siderophores associated with active growth have been reported in Azotobacter chroococcum (Page 1987; Suneja et al. 1994) and Bradyrhizobium Tal 1 000 (Carson et al. 1992). Complete inhibition of siderophore production at 5 11M iron concentration has been observed in Azospirillum lipoferum (Shah et al., 1993) and at 6 11M concentration in Azotobacter chroococcum (Suneja et al. 1994). However, in Rhizobium ciceri strain Ca181, siderophore production continued even at 500 11M concentration of iron, though the level of production decreased in other strains also. The decrease in hydroxamate type of siderophore varies from 30 to 75% at such high concentration of iron indicating that these strains continue to produce siderophores even in presence of sufficient levels of available iron. Total siderophore production determined by CAS assay also revealed production of siderophore at 500 11M concentration of iron in liquid medium though i1A-630 values were quite low in some strains (Fig. 3). Such weak responses or absence of response has been previously reported for Bradyrhizobium strains (Guerinot etal. 1990; Carson etal. 1992; Lesueuretal. 1993). Nine siderophore producers and one non-producer mutant were used for nodulation with chickpea cultivar C-235 to test whether siderophore production property could affect the nodulation and symbiotic effectivity. Although maximum plant dry weight, ARA per plant and total nitrogen per plant were observed with inoculation of strain Ca 181, this strain also produced maximum nodule fresh weight indicating clearly that higher effectivity of this strain was due to higher nodule biomass production. Ca181St4 produced maximum amount of hydroxamate siderophore and strain Ca181M115 produced maximum amount of total siderophores. In both the strains the relative nitrogen fixation potential was also higher which indicate involvement of siderophore in symbiotic nitrogen fixation. Similar results have been reported by O'Hara et al. (1988), Carson et al. (1992) and Van Rossum et al. (1994) in different Rhizobium spp. However, plants inoculated with strain Ca401RW5 (a non siderophore producer) also showed positive response to iron application in symbiotic nitrogen fixation and its effectivity in terms of total nitrogen per plant was comparable with strain Ca181. Van-Rossum etal. (1994) also observed high nodulation and nitrogen fixation by a non siderophore producing strain of Rhizobium sp. nodulating groundnut in presence of added iron. This could be due to direct iron available by the plants in iron sufficient conditions.
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©
Gustav Fischer Verlag
Role of siderophores in chickpea (Cicer arietinum L.) - Rhizobium symbiosis Monika Dhul, Sunita Suneja, K. R. Dadarwal Department of Microbiology, CCS Haryana Agricultural University, Hisar-125 004, India Accepted: January 18, 1998
Abstract Strains of Rhizobium ciceri (wild type as well as mutants) were screened for their ability to produce siderophores under cultural conditions. All strains except a mutant Ca401RW5 were found to be siderophore producers. A standard strain Ca 181 which is being used for large scale production as inoculant for chickpea, found to be a siderophore producer, was used for standardization of conditions for siderophore production. Only hydroxamate type of siderophores were detected under cultural conditions. The level of hydroxamate production increased linearly during log phase of growth up to 8 days. Addition of iron in the medium resulted in decrease in hydroxamate production and at 500 J.lM iron level, the hydroxamate level decreased by 40%. In other strains tested, decrease in hydroxamate production varied from 30 to 75% whereas in one strain Ca85AZ3 this level of iron completely inhibited hydroxamate production. Strain Ca 181 was most effective in terms of symbiotic nitrogen fixation in absence as well as added iron. Addition of iron stimulated nodule fresh weight in almost all the strains tested. It also resulted in increased symbiotic effectivity in terms of nitrogen gain per plant although the response varied with the bacterial strain. Key words: Rhizobium ciceri - siderophore - hydroxamate iron
Introduction Iron is essential for growth of all living organisms. Although, it is fourth most abundantly available element on the earth's crust but its availability to plants as well as microorganisms is hindered due to its easy chemical oxidation. It forms insoluble ferric salts, mainly hydroxides under acidic conditions and phosphates under neuCorresponding author: K. R. Dadarwal
tral to alkaline conditions. To solubilize and sequester iron, most of the microorganisms produce extracellular low molecular weight iron transport agents called siderophores (Neilands 1981) which are either phenolates or hydroxamates. Siderophore production in Rhizobium assumes special interest as iron is required not only for nodule formation but also for synthesis of key proteins required in nitrogen fixation viz. nitrogenase, hydrogen uptake hydrogenase and leghaemoglobin. How iron is obtained by the infecting bacterium inside the host system is not clear but it has been suggested that differences in nodule development under iron deficient conditions may be due to varying abilities of different strains of Rhizobium to acquire iron for nodule initiation and development (O'Hara eta!' 1988). Hydroxamate type siderophores are stable in soil over a wide range of pH and promote iron uptake by plants (Powell et a!. 1982; Reid et al. 1984). Scanty informations are available regarding production ofhydroxamate siderophores by rhizobia (Carson et al. 1992, 1994; Suneja eta!' 1992, 1994). Present study was carried out to investigate prevalence and nature of siderophores in strains of Rhizobium ciceri, the root nodule bacteria of chickpea (Cicer arietinum L.) to understand the role of siderophores in symbiosis. This micro symbiont is highly host specific.
Materials and methods Rhizobium strains. Rhizobium ciceri strains and some mutants of standard strains used in the present studies were procured from the Department of Microbiology, Chaudhary Charan Singh Haryana Agricultural UniverMicrobio!. Res. 153 (1998) 1
47
sity, Hisar. These strains were maintained by periodic transfer on Yeast extract mannitol agar slants (Vincent 1970). Screening of Rhizobium ciceri strains for siderophore production. The universal chemical assay as described by Schwyn and Neilands (1987) was used for qualitative detection of siderophores in different strains using chrome azurol S dye (CAS) agar plates for bacterial growth. A loopful each of 7d old cultures from Yema slopes was spotted on the medium plates and incubated at 30°C for 5 -7 d. Observations were taken for colony dia and halo zone formed around the colony to calculate halo zone area as described by Van-Rossum et al. (1994). Optimization of conditions. Optimization of conditions for maximum siderophore production with respect to culture media, stage of growth and iron concentration in the medium was done with Rhizobium ciceri strain Ca181. The strain was pregrown for 7 don YEMA slants and the cells from slants were suspended in sterilized saline. The cell suspension (0.1 ml) was used to inoculate 20 ml YEM broth (Vincent 1970) or Complete medium broth (Modi et al. 1985) in 50 ml flasks. The flasks were incubated at 300C on a rotary shaker for 4 d. The broth culture was then used to inoculate in fresh broth in multiple replications and incubated at 30°C on a rotary shaker. At different time intervals, three flasks of each medium were used for estimating protein and siderophores (hydroxamate and catechol type). Protein was estimated by the method of Lowry et al. (1951). The method of Csaky (1948) modified by Suneja and Lakshminarayana (1993) was used for estimation of hydroxamate type of siderophores and the method of Arnow (1937) was used for the estimation of catechol type of siderophores. Iron concentration. Optimum iron concentration was determined by growing the culture for 8 d in Complete medium (Modi et al. 1985) with different levels (0-500 11M) of iron in the form of FeCI3 . Siderophore production by different Rhizobium ciceri strains. The strains were grown in 20 ml of Complete medium broth without iron and with iron (500 11M FeCI3) in 50 ml conical flasks in triplicate and incubated at 30°C for 8 d on a rotary shaker. Protein and siderophores were estimated with appropriate methods described earlier. Total siderophores in culture supernatants after centrifugation were also estimated using chrome azurol S (CAS) assay method as described by Van-Rossum et al. (1994). Effect of iron on symbiotic effectivity. Healthy seeds of chickpea var C235 were surface sterilized with acidic alcohol (cone. sulphuric acid: ethanol, 7: 3, v/v) followed 48
Microbiol. Res. 153 (1998) 1
by thorough washing with sterilized water (Dadarwal et al. 1985). The surface sterilized seeds were inoculated with iron starved broth culture of ten selected Rhizobium isolates and their mutants. The inoculated seeds were sown in autoclaved chillum jar assemblies (Dahiya and Khurana 1981) containing washed river sand and Sloger's nitrogen free mineral salt solution (Sloger 1969) without iron and with iron. Uninoculated seeds were sown as controls. The jars were kept in a net house under day light conditions. The Sloger's nitrogen free mineral salt solution was modified to have 0 (control), 50 and 166 11M of FeCI 3• Thus for each strain two levels of iron with control (no iron) were kept to see the effect of added iron on symbiotic effectivity. The relative symbiotic effectivity of different strains was determined at 60 d of plant growth by taking into consideration the number of nodulus formed per plant, fresh weight of nodules, plant dry weight, nitrogenase activity and total plant nitrogen. Nitrogenase activity in nodules was determined by measuring acetylene reduction activity (Hardy et al. 1968). The nodules were detached from the roots after measuring acetylene reduction activity, counted and then weighed for fresh nodule weight. Total nitrogen content of the plants was determined calorimetrically using Nessler's reagent (Linder 1944).
Results Screening of Rhizobium ciceri strains for siderophore production Table 1 shows relative siderophore production by different strains of Rhizobium cieeri on CAS agar plates. All strains except a mutant Ca401RW5 showed production of siderophore. Strain CaI81-17-3B failed to grow on the CAS agar plates. Siderophore production was "corrected" for growth by dividing the halo zone area by the colony area (RIG; Table 1). Nine strains showed ratio between 1.7 to 3.2 which were considered to be low producers, 8 showed a ratio of 3.6 to 5.0 (moderate siderophore producers whereas 3 strains namely Ca534St8, Ca339 and Ca85St7 showed ratio more than 7.0 and were considered to be high siderophore producers. Effect of culture media and stage of growth on siderophore production Production of hydroxamate and catechol type of siderophore was studied in Rhizobium ciceri strain Ca181 to see the effect of medium composition and stage of bacterial growth. Catechol type of siderophore was absent at all stages of growth in both Complete and YEM medium. Maximum hydroxamate production was observed
Table 1. Relative siderophore production by different strains of Rhizobium ciceri on CAS agar plates Wild strain! mutant
Siderophore production Colony growth (G) (dia)
Halo zone (H) (dia)
Growth to halo zone ratio (G: H)
1.8 1.5 1.5 1.0 2.5 1.0 1.0 1.0 1.0 1.5 1.5 1.5 1.5 2.0 3.0 2.0 1.5 1.0 No growth 2.0 0.5 2.0
3.5 3.0 3.0 2.0 8.0 1.5 1.5 1.5 7.0 2.5 2.5 4.0 2.0 4.0 4.5 4.5 3.0 1.8
3.78 4.00 4.00 4.00 10.24i 2.25 2.25 2.25 49.00i 2.77 2.77 7.1 Ji 1.77 4.00 2.25 5.06 4.00 3.24
~
~
Table 2. Production of hydroxamate siderophore in Rhizobium ciceri strains as affected by iron* Wild Strain/ Mutant
Protein (/lg ml- I )
Hydroxamate (/lg Nmg- I protein)
FeFe FeFe-
93 146
6.56 2.95
222 267
1.39 0.93
CA 181 St4
FeFe+
145 170
8.62 2.82
CA 181 M 115
FeFe FeFe+
77 98
3.60 0.81
90 105
3.50 2.28
Ca 85 St 7
FeFe+
110 175
2.50 1.13
Ca339
FeFe
107 219
2.20 0.57
Ca410R
FeFe FeFe+
123 235
1.21 0.53
235 256
0.72
Ca401 RW 5
FeFe+
248 272
(-) (-) (-)
Ca534 St8
FeFe
205 230
7.81 5.34
G =Growth expressed as colony diameter (mm) H =Siderophore production expressed as halo diameter (mm) H/G =Halo area (0.5 H)2 divided by growth area (0.5 G)2 ~ =Not detected i =High value caused by small colony size
Ca32
FeFe
195 219
5.98 4.56
Ca 19 ANal
FeFe FeFe+
190 210
5.71 4.11
179 195
4.57 2.50
at the end of log phase on 8th d with corresponding production level of 5.7 and 1.8 flg N mg- I protein in Complete and YEM medium respectively (Fig. 1).
Ca534
FeFe+
172 185
3.65 2.40
Ca2 St9
FeFe
137 150
3.35 2.02
Effect of iron
CV4
FeFe+
130 141
2.53 1.88
Ca2
FeFe FeFe+
120 136
2.00 1.45
99 112
1.87 1.25
FeFe FeFe FeFe
87 105
(-) (-)
110 175
3.41 1.37
123 235
1.21 0.51
Ca 13 Ca32 Ca46 Ca 171 Ca339 CV4 Ca85 CA 85 Az 3 CA85 St7 Ca 85 St 10 Ca534 Ca 534 St 8 Ca2 Ca2 St 9 Ca181 Ca 181 M 115 Ca 181 St4 Ca 181-7-3B Ca 181-17-3B Ca40IR Ca40l RW5 Ca 19ANal
4.5
5.06
~
~
3.0
2.25
Fig. 2 shows effect of iron on hydroxamate siderophore production in strain Ca 181. Production of hydroxamate decreased with increase in concentration of iron in the medium. There was 40% decrease in production of hydroxamate at 500 flM iron concentration. Shah et ai. (1993) observed complete inhibition of hydroxamate siderophore in Azospirillum iipoferum at 5 flM concentration of iron.
Ca 181 CA 181-17-3 B
Ca85
Ca 85 Az 3
Ca13
Ca 181-17-3 B Ca46 Ca 85 St 10
Hydroxamate production in different Rhizobium ciceri strains
Ca171
Table 2 shows production of hydroxamate siderophores in Rhizobium ciceri strains. All strains except Ca401RW5 and Ca46 showed production of hydroxamate siderophores. In the presence of iron (500 flM), hy-
* Iron added in form of FeC1 3 (500 /lM) in Complete medium. Results are average of three replicates.
Microbial. Res. 153 (1998) 1
49
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Fig. 1. Effect of medium composition on bacterial cell protein and hydroxamate siderophore in relation to time of incubation. 0 Protein in Complete medium, [J [J Hydroxamate in Complete medium, 8 8 Hydroxamate yield in Complete medium, 0 ---- 0 Protein in YEM medium, [J [J Hydroxamate in YEM medium, 8 --- 8.Hydroxamate yield in YEM medium.
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Fig. 2. Effect of iron concentration on hydroxamate type of siderophore production in Rhizobium ciceri strain Ca 181 at 8 d of growth. -[J- Protein, -8- Hydroxamate yield. 50
Microbiol. Res. 153 (1998) 1
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Fig. 3. Siderophore production by Rhizobium ciceri strains in Complete medium with or without added Fe. and assayed with CAS supernatant assay (With shuttle). Table 3. Effect of inoculation of siderophore producers and non-producers strains of Rhizobium ciceri on symbiotic effectivity in chickpea cv. C 235 at 60 d under chillum jar conditions Inoculant Strain
Nodule No. (pi-I)
Nodule fresh weight Shoot dry weight (mg pi- I) (mg pi- I)
ARA (n moles h- I pi- I)
Total plant nitrogen (mg pi-I)
Fe- Fe 1 Fe2
Fe-
Fe-
Fe-
Fe 1 Fe2
Control Ca 181
19
20
2
11
4
28
27
Cal 181-17-3 B 5
10
9
Ca 181 M 115 Ca 181 St4
322
Fe-
Fe 1
Fe2
229
265
319
503
483
361
371
478
12
158
267
272
298
103
296
257
283
371
368
15.64
9
29
137
109
279
319
295
5.95
17
112
114
136
275
288
323
11.05
36.04
Ca85
7
Ca 85 Az 3
3
13
14
34
164
186
244
266
388
53.40
Ca 85 St 7
7
12
13
112
148
164
251
287
327
8.87
Ca339
2
3
3
29
32
35
265
353
389
15 .51
Ca401 R
5
8
11
85
123
226
261
278
289
7.66
Ca401 RW 5
2
18
24
20
189
263
263
324
427
9
Fe 1
Fe2
4.1 (1.8) 114.84 179.20 8.2 (2.4) 7.15 10.46 5.8 (2.2) 27.20 45 .33 6.2 (2.2) 7.05 37 .05 5.3 (1.9) 20.07 43 .71 6.6 (2.4) 55.63 55.63 4.8 (2.0) 48.50 50.20 5.7 (2.3) 28.60 33.11 5.5 (2.1) 49.67 66.83 5.7 (2.2) 21 .53 59.71 5.5 (2.1)
Fe 1
Fe2
5.6 (2.1) 9.2 (2.5) 7.6 (2.8) 8.9 (2.4) 7.0 (2.2) 7.2 (2.5) 7.1 (2.7) 7.1 (2.5) 8.4 (2.4) 6.9 (2.5) 8.1 (2.5)
7.0 (2.2) 11.9 (2.5) 8.3 (2.9) 9.5 (2.6) 8.2 (2 .8) 8.7 (2.7) 10.8 (2.8) 8.1 (2.5) 9.7 (2.5) 7.2 (2.5) 10.6 (2.5)
Fe- - Iron absent; Fe 1 - 50 flM iron; Fe 2 - 166 flM iron. Figures in parentheses indicate % N. Values are average of three replicates. Microbiol. Res. 153 (1998) I
51
droxamate level decreased approximately from 30 to 75% in different strains. In strain Ca85Az3 this level of iron completely inhibited hydroxamate production. Above strains were further tested for total siderophore production using CAS supernatant assay with shuttle. All strains except Ca401RW5 were found to be siderophore positive (Fig. 3). The responses of many tested strains were relatively weak i.e. small b.-A 630 values. Symbiotic effectivity of Rhizobium ciceri strains under chillum jar conditions
Table 3 shows effect of inoculation of siderophore producers and nonproducer strains on symbiotic effectivity in chickpea cv 235. Nodulation, plant dry weight and total nitrogen per plant were more in treatments in which iron was used than control without iron thus clearly showing the role of iron in symbiotic nitrogen fixation. Maximum plant dry weight, ARA per plant and total nitrogen per plant was observed with inoculation of strain Ca181. This strain also produced maximum nodule fresh weight indicating clearly that higher effectivity of this strain was due to higher nodule biomass production. Other strains which responded to iron with regard to gain in total plant nitrogen were Ca181St4, Ca181M115, Ca181-17-3B and Ca85Az3. Strain Ca181St4 produced maximum amount of hydroxamate siderophore and strain Cal81M115 produced maximum amount of total siderophores. In both the strains the relative nitrogen fixation potential taking into consideration the relative nodule biomass was also higher which indicate involvement of siderophore production in symbiotic nitrogen fixation.
Discussion Detection of siderophores using chrome azurol S (CAS) agar plate is based on the ability of siderophore to act as chelator with varying affinity for iron. The presence of this iron chelator is indicated by decolorization of blue coloured ferric CAS complex, resulting in an orange halo around the colonies on CAS plates. In the present study, this approach was followed and 22 strains and mutants of Rhizobium ciceri were screened for siderophore production, out of which 20 were found to be positive, one was negative and one showed no growth on CAS agar plate. YEM medium and Complete medium were used for detection of hydroxamate or catechol type of siderophores in Rhizobium ciceri strain Ca181. Catechol type of siderophore was absent in this strain but the strain produced hydroxamate type of siderophore which was maximum at the end of log phase. Absence of catechol type of siderophores has been reported earlier also in dif52
Microbial. Res. 153 (1998) 1
ferent species of Rhizobium (Carson et al. 1992; Suneja et al. 1992). These workers found production of hydro xamate type of siderophores in the rhizobia studied. Similar to our observations production of siderophores associated with active growth have been reported in Azotobacter chroococcum (Page 1987; Suneja et al. 1994) and Bradyrhizobium Tal 1 000 (Carson et al. 1992). Complete inhibition of siderophore production at 5 11M iron concentration has been observed in Azospirillum lipoferum (Shah et al., 1993) and at 6 11M concentration in Azotobacter chroococcum (Suneja et al. 1994). However, in Rhizobium ciceri strain Ca181, siderophore production continued even at 500 11M concentration of iron, though the level of production decreased in other strains also. The decrease in hydroxamate type of siderophore varies from 30 to 75% at such high concentration of iron indicating that these strains continue to produce siderophores even in presence of sufficient levels of available iron. Total siderophore production determined by CAS assay also revealed production of siderophore at 500 11M concentration of iron in liquid medium though i1A-630 values were quite low in some strains (Fig. 3). Such weak responses or absence of response has been previously reported for Bradyrhizobium strains (Guerinot etal. 1990; Carson etal. 1992; Lesueuretal. 1993). Nine siderophore producers and one non-producer mutant were used for nodulation with chickpea cultivar C-235 to test whether siderophore production property could affect the nodulation and symbiotic effectivity. Although maximum plant dry weight, ARA per plant and total nitrogen per plant were observed with inoculation of strain Ca 181, this strain also produced maximum nodule fresh weight indicating clearly that higher effectivity of this strain was due to higher nodule biomass production. Ca181St4 produced maximum amount of hydroxamate siderophore and strain Ca181M115 produced maximum amount of total siderophores. In both the strains the relative nitrogen fixation potential was also higher which indicate involvement of siderophore in symbiotic nitrogen fixation. Similar results have been reported by O'Hara et al. (1988), Carson et al. (1992) and Van Rossum et al. (1994) in different Rhizobium spp. However, plants inoculated with strain Ca401RW5 (a non siderophore producer) also showed positive response to iron application in symbiotic nitrogen fixation and its effectivity in terms of total nitrogen per plant was comparable with strain Ca181. Van-Rossum etal. (1994) also observed high nodulation and nitrogen fixation by a non siderophore producing strain of Rhizobium sp. nodulating groundnut in presence of added iron. This could be due to direct iron available by the plants in iron sufficient conditions.
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