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Soil Biology & Biochemistry 46 (2012) 115e122

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Soil Biology & Biochemistry

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Calcium affects the competitiveness of acid-sensitive and acid-tolerant strainsof Bradyrhizobium japonicum in nodulating and fixing nitrogen with twosoybean cultivars in acid soil

Arief Indrasumunar a,b,c, Neal W. Menzies b, Peter J. Dart b,*a Indonesian Centre for Agricultural Biotechnology and Genetic Resources Research and Development, Bogor 16111, Indonesiab School of Agriculture and Food Sciences, The University of Queensland, St. Lucia 4072, AustraliacARC Centre of Excellence for Integrative Legume Research, The University of Queensland, St. Lucia 4072, Australia

a r t i c l e i n f o

Article history:Received 12 April 2011Received in revised form20 July 2011Accepted 14 November 2011Available online 9 December 2011

Keywords:BradyrhizobiumAcid-sensitiveAcid-tolerantSoybeanCompetitivenessNodulationHost cultivar Bradyrhizobium interaction

Abbreviations: ATR, acid-tolerance response; YMA* Corresponding author. Tel.: þ61 7 33652867; fax:

E-mail addresses: p.dart@uq.edu.au, pjdart@gmail

0038-0717/$ e see front matter � 2011 Elsevier Ltd.doi:10.1016/j.soilbio.2011.11.017

a b s t r a c t

A glasshouse experiment studied the role of calcium and pH on competitiveness of acid-sensitive andacid-tolerant Bradyrhizobium japonicum strains with similar N2-fixation effectiveness in nodulating twosoybean (Glycine max L. Merr) cultivars selected for tolerance of aluminium (PI416937) or for manganese(Manta). Liming provides calcium (Ca) as well as increasing soil pH. Thus the effect of Ca and pH of soilare difficult to separate. We examined the effects of Ca per se by comparing the response to gypsum andlime amendment on the competitiveness of acid-tolerant and acid-sensitive strains in nodulatingsoybean in an acid soil. Acid soil was treated with either CaSO4 or CaCO3 and incubated for 2 weeksbefore sowing soybean seed. Two acid-sensitive and two acid-tolerant B. japonicum strains were mixedwith each other (one acid-sensitive plus one acid-tolerant) and were inoculated onto soybean seeds atthe rate of 106cfu seed�1. Soil pH, as amended by lime addition, had more effect on nodulation than Caaddition in the form of gypsum. The response was affected by cultivar and strain in a complicated fashionwith a marked strain � cultivar interaction. One acid-tolerant strain formed most nodules with bothcultivars in the unamended soil of pH 4.36 in competition with one acid-sensitive strain. The same acid-tolerant strain was not competitive against the second acid-sensitive strain with Manta but was withPI416937. The second acid-tolerant strain was not competitive with either acid-sensitive strain inunamended and gypsum treated soils. It was only competitive with PI416937 in limed soil, a rathersurprising result. Inoculation of this soil with no native soybean nodulating strains, increased shootweight, %N, N uptake. N2-fixation was greatly increased by inoculation and lime addition, and to a lesserextent by gypsum addition for Manta. This experiment indicates that addition of Ca per se as gypsum toan acid soil has little effect on symbiotic performance, but changing pH by liming has a major effect, thatboth soybean cultivar and B. japonicum strain influence the competitiveness of strains in acid soil andthat acid-tolerance does not necessarily increase a strain’s competitiveness.

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1. Introduction

The main criterion used in the selection of strains of Bradyrhi-zobium for legume inoculation is the ability to form an effectivesymbiosis with the hosts for which the inoculant is recommended.However, inoculation may not lead to improved nodulation orenhanced N2-fixation because of the presence of indigenous rhizobiawhich are more competitive than the inoculant strain (Roughleyet al., 1976). Therefore, Brockwell et al. (1968) suggested that the

, yeast mannitol agar.þ61 7 33651177..com (P.J. Dart).

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ability to persist in soil, and to compete, should be included in thecriteria used to select improved inoculant strains. Any strain witha combination of competitiveness, persistence and N-fixing potentialwould ensure better and consistent performance. This implies theneed to either improve the competitiveness of a strain characterizedfor effective N2-fixation, or improve the N2-fixing capacity ofa competitive strain. Alternatively, the effective isolates could bescreened for competitiveness. This would require an easily identifi-able marker for the inoculant strain that does not have a plei-omorphic effect on other symbiotic competence factors.

Competition among Rhizobium strains for nodulation of theirlegume host is an important aspect of the ecology of the root nodulebacteria. The mechanisms that confer competitive advantage to

A. Indrasumunar et al. / Soil Biology & Biochemistry 46 (2012) 115e122116

a strain are poorly understood. Recent research indicates that hostspecificity of rhizobia has an underlying genetic mechanism that issimilar to pathogenic bacteriaehost interactions (Yang et al., 2010)and the modulation of successful infection as expressed throughcompetitiveness may be related to subtle variations in plant resis-tance (R) genes and rhizobial surface polysaccharides (Jones et al.,2008). Since competitiveness is crucial for the success of inocula-tion, manipulation of the association through strain selection (e.g.McLoughlin et al., 1990), plant selection (e.g. Keyser and Cregan,1987), modification of inoculation rate (Weaver and Frederick,1974a, 1974b; Reyes and Schmidt, 1981) and inoculation method(Boonkerd et al., 1978) have been attempted to improve infection.

Soil pH affects the competitive ability of Rhizobium trifoliiserogroups (Dughri and Bottomley, 1983) and Rhizobium legumino-sarumbv. phaseoli strains (FreyandBlum,1994). Acid-tolerant strainsformed more nodules than acid-sensitive strains in unlimed acidicsoil, whereas in limed soil the acid-sensitive strainwas the dominantnodule occupant (Frey and Blum, 1994). Calcium (Ca) modified thepH effect on the growth of acid-tolerant and acid-sensitive strains ofRhizobium meliloti in acidic media (Howieson et al., 1992). Growthrate ofR.melilotiwas increased by increasingCa concentration at lowpH, but not at neutral pH, so, requirement for Ca at low pH is greaterthan at neutral pH. However, the attempts to find why the nodula-tion ability of R. meliloti is so poorly tolerant of acidity in the plantgrowth media have been unsuccessful. Although the poor nodula-tion of Medicago spp. is related to interaction of specific signalmolecules between plant and rhizobia it is not due to a change inproduction of root exudate inducers of the nodB genes (Watkin et al.,2009). For soybean (Glycine max L. Merr) and bean (Phaseolus vul-garis) Hungria and Stacey (1997) found that plant nod-gene inducerproductionwas less at pH 4.5 than at 5.8. However, the acid-tolerantRhizobium tropici strain CIAT 899 produces more and different Nodfactors at pH 4 than at pH 7, an adaptation to acidic soil conditions(Moron et al., 2005). Clearly the role of pH on signal interactions innodulation may well differ between plant species which are nodu-lated by quite different rhizobial species. The promise of adding nodgene inducers to inoculants to enhance nodulation in stressful soilconditions, such as low pH, has not yet materialized as a practicalfield process.

An acid-tolerance response (ATR) has been induced in somerhizobial strains of Sinorhizobiummeliloti (e.g. Draghi et al., 2010) andMesorhizobium spp. and Mesorhizobium huakuii (e.g. Rickert et al.,2000) by growing the strains in a mildly acid medium before sub-jecting them to amore acidic environment. Such an ATR response forS. meliloti resulted in an increased competitiveness in nodulation oflucerne (Medicago sativa) for the adapted strain in sterile vermiculitemedium. It remains to be seen if this translates to a field soilnodulation response and increased N2-fixation, and whether an ATRprocess occurs when rhizobia grow naturally in acid soils or migrateafter inoculation from the rhizosphere to the bulk soil.

The effects of aluminium (Al) and proton (Hþ) toxicity and Cadeficiency in acid soils are difficult to separate in field experimentsbecause liming provides Ca as well as increasing the pH of the soil.Thus, lime responses of legumes have been attributed to alleviationof Ca deficiency (Russell, 1966), increased Ca availability andreduction in soluble Al (Isbell et al., 1976; Fox et al., 1985). Thepresent study examines the effects of pH and Ca on competition fornodule occupancy of acid-tolerant soybean cultivars by acid-tolerant and acid-sensitive strains in an acidic soil with no indig-enous rhizobia. Further we examine the effect of pH and Ca addi-tion on N2-fixation as it is the amount the nodules fix that is theimportant agronomic outcome. We investigate the effects of Caaddition per se on symbiotic performance through comparinggypsum and lime addition for the provision of the Ca. Lime alsochanges pH but gypsum has a small effect on pH.

2. Materials and methods

2.1. Soil treatments

The topsoil used was collected from a texture contrast Alba-quult (USDA and NCRS, 2006) (Yellow Kurosol, Isbell, 2002). Thesoil amendments, lime (CaCO3) and gypsum (CaSO4$2H2O), wereused to manipulate the soil pH and Ca status. Incubation of soilsamples (200 g) with a range of lime additions was used toestablish that an addition of 2.00 g of CaCO3 kg�1 was required toincrease the soil pH from 4.36 to 5.50. When gypsum is added tosoil, the pH typically decreases as a result of the increased soilsolution ionic strength (Menzies et al., 1991). To counteract this pHdecrease, 0.40 g CaCO3 kg�1 soil was required in gypsum treatedsoils. On the basis of these results, the soil treatments applied were,Control (no added calcium), Gypsum (2.73 g CaSO4$2H2O þ 0.40 gCaCO3 kg�1 soil), and Lime (2.00 g CaCO3 kg�1 soil). The amount ofCa added in the gypsum and lime treatments was equivalent. It wasenvisaged that these treatments would permit discriminationbetween Ca and pH effects on strain competitiveness for nodula-tion. The amendments were well mixed through the soil in bulkusing a cement mixer. The 15 cm diameter pots contained 1500 gair dry soil. Pots were then watered to soil field capacity andincubated for 2 weeks. Soil pH (1:2.5 H2O) was measured after theincubation period.

On the basis of soil analysis, this soil was deficient of phosphorusand potassium, so basal fertilizer as KH2PO4 was applied in solutionat the rate of 100 kg P ha�1 and 126 kg K ha�1 calculated on a soilweight basis (bulk density 1.33 g cm�3).

2.2. Inoculant preparation

Four strains, two acid-tolerant that did not have any intrinsicresistance to streptomycin antibiotic (FCB34 and FCB206), and twoacid-sensitive that were selected for resistance to streptomycin500 mg mL�1 (FCB179 and FCB146), were grown in 125 mL yeastmannitol (YM) broth in a 250 mL flask. These strains were isolatedfrom nodules of soybean plants grown at various sites in Indonesia.The streptomycin mutant strains were naturally-occurring mutantsselected from a wild type strain population selected by Mr. GregGemell (NSW Department of Primary Industries Gosford PrimaryIndustries Institute, Narara). Three strains (FCB146, FCB206, FCB34)were equivalent in their effectiveness in promoting plant growthwith soybean at neutral pH in sand culture and FCB179 was supe-rior and equivalent to commercial strain CB1809 (Indrasumunar,1999). After 7 days incubation at 28 �C on a rotary incubator-shaker (150 rpm), the total number of bradyrhizobia for eachstrain was counted using the drop plate method. Viable cellnumbers (expressed as log10) were, FCB34 8.69 � 0.19, FCB2068.72 � 0.13, FCB146 8.86 � 0.08 and FCB179 8.98 � 0.20 (mean of 6replicates). Broth cultures were then mixed and diluted with sterile¼ strength Fahraeus media (Fahraeus, 1957) to bring the celldensity to around 105 cells mL�1. The number of viable cells ofdiluted broth culture of each strain was determined by drop plateand spread plate count onto yeast mannitol agar (YMA) and YMAcontaining streptomycin 500 mg mL�1. Suspensions of paired acid-tolerant and acid-sensitive strains were prepared in ¼ Fahraeusmedia in combinations and numbers as shown in Table 1.

2.3. Plant preparation

Soybean cv. PI416937 (Al-tolerant) and Manta (Mn-tolerant)seedswere surface sterilized bywashing in alcohol for 30 s followedby soaking in 1.2% HClO4 for 5 min. After 7 separate rinses in sterilewater the seed was soaked for 2 h in the final rinse. Five seeds were

Table 2Changes in soil pH (1:2.5 H2O) during soybean growth. Average of 3 potmeasurements.

Soil treatment Day 0 Day 14 Day 36

Control 4.36 4.27 3.93Gypsum 4.42 4.39 4.06Lime 5.75 5.28 4.93

Table 1Mean log10 number of B. japonicum for each strain after dilution and mixing, and theeffect of inoculation treatments on the nodule number of soybean at day 40. Nodulenumber data with the same letters are not significantly different at the 1% level ofsignificance. Average of 6 replicates.

Combination treatment,Tolerant � sensitive

Mean log10 cells seed�1 Nodule numberper plant

Tolerant Sensitive

(i). FCB34 � FCB179 5.05 � 0.09 5.25 � 0.15 16.4b

(ii). FCB34 � FCB146 5.02 � 0.08 5.12 � 0.13 11.2c

(iii). FCB206 � FCB179 5.20 � 0.11 5.22 � 0.10 17.1a

(iv). FCB206 � FCB146 5.16 � 0.14 5.09 � 0.13 20.0a

Uninoculated e e 0d

A. Indrasumunar et al. / Soil Biology & Biochemistry 46 (2012) 115e122 117

sown into each 15 cm dia pot. Three replicates were prepared foreach treatment. Soybean seeds were inoculated with 1 mL of theappropriate suspension (Table 5). Treatments were randomized inblocks in a glasshousewith a day/night temperature of c. 33 �C/28 �Ccontrolled by evaporative cooling. After germination, plants werethinned to three per pot, before adding white, sterile, plastic beadsto the soil surface to reduce aerial cross-contamination and mois-ture loss. Pots were watered daily through a watering tube to fieldcapacity after weighing to determine water loss. Plants were har-vested at 40 days after planting, root nodules were separated,washed and kept in a freezer (�20 �C) before occupant strainidentification. Shoot and root dry-weight, and nodule number andfresh weight were determined. The statistical analysis was per-formed as a 2 � 3 � 5 factorial (soybean cultivar � soil treat-ment � inoculation treatment) with three replications ina completely randomised block design.

2.4. Soil properties

Soil pH was measured during plant growth at day 0 (beforeinoculation),14, 36 and 40 (harvest time). After harvesting, soilswerecarefully separated from roots and nodules, and soil solution wasextracted by centrifuge drainage (Gillman, 1976). Soil solutions werefiltered to 0.22 mm (Millipore GSWP) and a portion was furtherfiltered to 0.025 mm (Millipore VSWP) to avoid possible contamina-tion of the filtered solution by Al-containing particulates (Menzieset al., 1991). Soil solution pH and electrical conductivity was deter-mined on 0.22 mm filtered solution, and inorganic monomeric Al3þ

concentration (Kerven et al., 1989) on 0.025 mm filtered solutionwithin8hof extraction. The remaining0.025mmfiltered solutionwasstored at 4 �C for later elemental analysis by inductively coupledplasma atomic emission spectroscopy. Solution speciation wascalculated using GEOCHEM (Sposito and Mattigod, 1980).

2.5. Nodule typing

Nodules were surface sterilized by immersion in 95% ethanol for10 s and HClO4 (1.2%) for 5 min. They were thenwashed thoroughlywith at least seven changes of sterile deionized water. Each nodulewas crushed in a small drop of sterile deionized water with a sterileorange stick and the suspension patched continuously onto a YMAplate, YMA containing streptomycin 500 mg mL�1, and a secondYMA plate (Vincent, 1970).

Inoculation with a single strain was used as a control to checkthe validity of the identification methods that were applied inthis experiment. Rhizobia from squashes of nodules formed bysoybean inoculated with a strain with no antibiotic resistance(e.g. FB34 or FB206) did not grow in the antibiotic media(500 mg g�1 streptomycin). On the other hand, all nodulesquashes of soybean nodules formed by inoculation with natu-rally occurring antibiotic resistant strains were able to grow on

antibiotic media (500 mg g�1 streptomycin). These results showedthat strain identification in nodules in our competition experi-ments could be based on plating nodule squashes on agar mediawith and without antibiotic. In addition, each strain used in thisstudy had a different colony appearance on YMA medium con-taining congo red dye. The colony appearance of acid-tolerantstrains that did not have any antibiotic resistance were raisedgummy reddish (FCB34), and high raised pinpoint cream/pink(FCB206). On the other hand, acid-sensitive strains that wereresistant to 500 mg mL�1 streptomycin had a different colonyappearance of raised pinpoint cream (FCB146), and high raisedpinpoint cream/white (FCB179). We thus had a double check toreliably confirm which strain occupies each nodule (antibioticresistance and colony appearance).

Statistical analysis was conducted using SAS programs (Anon,1999) for analyses of variance and Duncan’s multiple range testsof difference between treatments at the 5% significance level.

3. Results

3.1. Treatment effects on soil conditions and plant growth

The soil treatments were intended to achieve a range of Ca andAl status in the soil solution. Lime application was intended toraise the soil pH and hence lower the Al concentration, while theapplication of a small amount of lime in the gypsum treatmentwas intended to maintain the soil pH close to that of the controltreatment. Thus the gypsum treatment would maintain a similarlevel of Al challenge as that presented by the control soil, butincrease the soil solution Ca concentration. After 2 weeks incu-bation, the addition of gypsum (þlime) resulted in a smallincrease in soil suspension pH (0.06 pH unit), while the addition oflime resulted in an increase of 1.39 pH units (Table 2, Day 0).During the soybean growth, the pH of all treatments decreasedslightly, but the relative differences between the treatments weremaintained (Table 2).

The soil solution composition provides a more detailedimpression of the Ca and Al status of the root environment. Limetreatment markedly increased the soil solution Ca concentration,raised the pH and lowered the Al and Mn concentration (Table 3).The gypsum treatment caused a small increase in soil solution pH,and this is reflected in a loweredmonomeric Al3þ activity. However,the large increase in SO4 concentration resulted in an increasedtotal soil solution Al concentration, through Al-sulfate ion pairing.Gypsum increased the soil solution Ca concentration, but had noeffect on solution Mn concentration.

Lime and gypsum treatments both significantly (P < 0.05)increased shoot dry-weight, the lime treatment increasing yield byapproximately 50% relative to the control, while gypsum treatmentincreased yields by approximately 25% (Fig.1). The increase of shootdry-weight in the lime treatment is attributed to the increased soilpH and the reduced concentration of Al and Mn (Tables 2 and 3),promoting increased N2-fixation (Table 4). The gypsum treatmentyield increase may be attributed to the beneficial effect of Ca inreducing the Al toxicity, and to the reduction in the activity ofmonomeric Al3þ.

Table 3The effect of soil treatments on soil solution properties at day 40. Average of soil solution from 3 pots.

Soil treatment Caa mM Mga mM Ka mM Naa mM Pa mM Sa mM Total Ala mM Mono. Ala mM Mna mM pHb

Control 1900 2130 159 11900 2.58 312 452 63 162 3.77CaSO4 12900 1970 115 8890 2.90 12400 560 31 169 4.09CaCO3 5200 703 113 8100 0.97 571 14 11 7 5.01

a Determined on soil solutions filtered through 0.025 mm.b Determined on soil solutions filtered through 0.22 mm.

A. Indrasumunar et al. / Soil Biology & Biochemistry 46 (2012) 115e122118

3.2. Nodulation

No nodules were found on uninoculated plants; therefore, allnodules in the inoculated plants were formed by the inoculumstrains. There was a significant (P < 0.01) difference between straincombinations in nodule number (Table 1). Strain combinations (i),(iii) and (iv) formed 16, 17 and 20 nodules per plant (mean acrosstreatments), significantly (P < 0.001) more nodules than combina-tion (ii) (11 nodules per plant).

There was a significant interaction between soil treatments andsoybean cultivars in determining nodule number and fresh weight(Table 4). Liming significantly increased nodule number and freshweight with both soybean cultivars. Consistent with the Mn toler-ance of Manta (Rose, 1986), gypsum treatment did not increasenodule number in either cultivar, but increased nodule freshweightin Manta while with cv PI416937, gypsum treatment even hadslightly smaller nodule number and fresh weight than the no Caaddition control though not significantly (P < 0.01) so. In Manta,there was nodule size compensation in the gypsum treatment.Although nodule number was equal to the no added Ca, nodulefresh weight per plant was significantly (P < 0.01) larger. Mantaformed significantly (P < 0.01) more nodules (24 nodules plant�1)than PI416937 (9 nodules plant�1).

The detrimental effects of soil acidity on nodulation have beenattributed to the direct effect of pH or Hþ concentration, indirecteffects, such as toxicities of Al and/orMn, anddeficiencies of Ca, P andMo (Andrew, 1978). In the present experiment, soil pH (lime treat-ment) had significantly more effect on soybean nodulation than Calevel (gypsum treatment) (Table 4). The application of lime was ableto reduce acidity and toxicities of Al and/orMn aswell as increase theavailability of Ca, while the application of gypsum increased theavailability of Ca and S, but only caused a slight reduction in mono-meric Al3þ (Table 3).

There are beneficial effects of external Ca in reducing Al uptake(Huett and Menary, 1980) and of gypsum in improving plant growth

Fig. 1. Interaction between inoculation treatment (uninoculated (UI) and strain combi-nations i, ii, iii and iv) and soil treatment on shoot dry-weightof soybean at day 40. *valueswith the same letter are not significantly different at the 5% level of significance.

on Al toxic soils (Sumner et al., 1986), but this only happened in lowconcentrations ofmonomeric Al3þ (Brady,1991). Brady found that Caconcentration �150 mMwas able to improve growth and nodulationof soybean exposed to an Al activity of 7 mM. However, even anexternal Ca concentration of 2000 mM could not overcome the effectof 20 mM Al on soybean growth and nodulation. In the presentexperiment, increasing Ca concentration up to 13 mM (gypsumtreatment) could not overcome the effect of Al stress factors onsoybean PI416937 nodule formation and development. However, aswill be shown later, it did have a large effect on strain competition,favouring the acid-sensitive strain in both cultivars. It seems there-fore that Ca had a slight effect in reducing Al toxicity in noduledevelopment of soybean cv. Manta but not in the PI416937.

3.3. Nodule effectiveness

There was a significant (P < 0.05) interaction between soil treat-ments and inoculation treatments on shoot dry-weight (Fig.1). Therewas no significant (P < 0.05) difference between inoculation treat-ments with no added Ca, but in the gypsum and lime treatmentstherewere differences among inoculation treatments. In the gypsumtreatment, inoculationwith strain combination (i) (where nodulationwas dominated by the acid-sensitive strain FCB179) resulted in lessshoot dry-weight than for the strain combinations (iii) or (iv) (wherenodulationwasdominated by the acid-sensitive strain FCB146) or theuninoculated treatments. For the lime treatment, inoculationwith allstrain combinations increased shoot dry-weight compared to theuninoculated treatment or the other soil treatments. Comparingcultivars, Manta had significantly (P < 0.01) larger shoot dry-weight(1.69 g/plant) than for the PI416937 (2.04 g/plant) presumablyreflecting that Manta formed more nodules and nodule weight perplant.

On the basis of shoot dry-weight, there was no significant(P < 0.05) difference between inoculated plants and uninoculatedplants (Fig. 1) for 8 of the 12 inoculation treatment combinations,but on the basis of shoot N content, inoculated plants had signifi-cantly larger shoot N percentage and N content in all soil treat-ments (Table 5). The difference in shoot N percentage and N contentbetween inoculated and uninoculated plants was much larger inthe lime treatment than for the control and gypsum treatments. Inthe limed treatment the difference in N content plant�1 was41.5 mg, while in the gypsum treatment it was 15.0 mg and for theno added Ca treatment it was 11.2 mg.

Table 4Interaction between soil treatments and soybean cultivars on nodule number,nodule fresh weight, and presumptive N2-fixation at day 40. For each parameter,values with the same superscript letter are not significantly different at the 1% levelof significance.

Treatment Nodule number Nodule freshweight (g/plant)

Presumptive N2-fixation (g/plant)

PI416937 Manta PI416937 Manta PI416937 Manta

Control 7.4c 18.5b 0.085d 0.218c 0.008e 0.014d

Gypsum 3.9c 17.9b 0.065d 0.316b 0.006e 0.023c

Lime 14.8b 34.6a 0.363b 0.497a 0.034b 0.048a

Table 5Effects of inoculation and soil treatments, or soybean cultivar, on shoot N concen-tration and content at day 40. For each parameter, values with the same superscriptletter are not significantly different at the 1% level of significance.

Shoot N concentration (%) Shoot N content (g/plant)

Inoculated Uninoculated Inoculated Uninoculated

Soil TreatmentControl 3.4a 2.5c 0.048b 0.037c

Gypsum 2.7b 2.0d 0.053b 0.038c

Lime 3.4a 1.9d 0.082a 0.040c

Soybean cultivarPI416937 3.2a 2.3b 0.053b 0.037c

Manta 3.2a 1.9c 0.069a 0.040c

A. Indrasumunar et al. / Soil Biology & Biochemistry 46 (2012) 115e122 119

There were also significant (P < 0.01) differences betweensoybean cultivars in shoot N percentage and N content. Soybeancultivar PI416937 had a greater leaf N concentration (2.71%) than forManta (2.56%), but significantly (P< 0.01) lower N content (PI4169370.045 g/plant, Manta 0.055 g/plant) because Manta had larger shootdry-weight than PI416937.

There was a significant (P < 0.01) interaction between soybeancultivar and inoculation treatment on N percentage and N content(Table 5). In both parameters Manta had larger differences betweeninoculated and uninoculated plants. This is a good indication thatManta had fixed more N2 than PI416937.

The amount of N2 fixed was estimated by the N differencemethod, in which the N yield of the shoot of uninoculated soybeanwas subtracted from that of the inoculated soybean and thedifference assumed to be derived from N2-fixation. PresumptiveN2-fixation in Manta was significantly (P < 0.01) larger than forthe PI416937 (Table 4). In both soybean cultivars, liming signifi-cantly (P < 0.01) increased N2 fixed. In PI416937, there was anincrease of 26 mg plant�1 (4-fold); while in Manta the increasewas around 34 mg plant�1 (3.5-fold) over that for plants notreceiving lime. Gypsum application did not increase the N2 fixedin PI416937, but in Manta the amount of N2 fixed increased byaround 10 mg plant�1. This probably reflects the tolerance ofManta for Mn cf. PI416937.

There was a good correlation between nodulation, N contentand presumptive N2-fixation. The gypsum treatment that did notincrease nodulation of PI416937 (Table 4) also did not increase N2-fixation (Table 4) of this cultivar. On the other hand, gypsumtreatment likewise did not increase nodule number on Manta, butdid increase nodule fresh weight and N2-fixation. Similarly, Mantathat had significantly larger nodule fresh weight (Table 4) also hada larger N2-fixation than PI416937.

3.4. Strain competitiveness

The patterns of competition between acid-tolerant and acid-sensitive strains are given in Fig. 2. For both soybean cultivars, theacid-tolerant strain (FCB34) was less competitive than both the acid-sensitive strains [FCB179 (Fig. 2a) and FCB146 (Fig. 2b)]. In combi-nation (i) (FCB34 � FCB179) and combination (ii) (FCB34 � FCB146),the acid-tolerant strain (FCB34) did not form any nodules on Manta.In PI416937, the addition of gypsum and lime increased thecompetitiveness of the acid-tolerant strain (FCB34).When FCB34waspaired with the acid-sensitive FCB179, the addition of gypsum andlime slightly increased nodule occupancy of the acid-tolerant strain,but this strainwas still much less competitive than the acid-sensitivestrain. In contrast, when FCB34 was paired with the acid-sensitivestrain FCB146, the addition of lime increased the competitivenessof the acid-tolerant strain resulting in it formingmore than90%of thenodules.

The competition patterns between the acid-tolerant strain(FCB206) and the acid-sensitive strains [FCB179 (Fig. 2c) and FCB146(Fig. 2d)] were affected by both soil treatment and soybean cultivar.When FCB206 was paired with the acid-sensitive strain FCB179(Fig. 2c), soil treatment affected the pattern of strain competitivenessin PI416937 but not in Manta. For PI416937 with no added Ca, theacid-sensitive strain was out competed by the acid-tolerant strain aspredicted. The addition of gypsum and lime improved the compet-itive ability of the acid-sensitive strain so that it became dominant innodule occupancy. In Manta, the acid-sensitive strain FCB179 wasvery dominant, forming all nodules tested in all soil treatments. Soiltreatment did not change the pattern of strain competitiveness inthis cultivar. However, when FCB206 was paired with the other acid-sensitive strain (FCB146) (Fig. 2d), the acid-tolerant strain dominatedsoybean nodulation in the control treatment with no added Ca. InPI416937 the acid-tolerant strain (FCB206) occupied 95% of thenodules in the control treatment, while in Manta it occupied 97% ofthe nodules. Addition of Ca as gypsum increased competitiveness ofthe acid-sensitive strain in both soybean cultivars so that inPI416937, it occupied 79% of the nodules, while inManta, it occupiedall nodules tested. There was a difference in competition patternbetween soybean cultivars in response to liming. In PI416937, thenodules were dominated by the acid-tolerant strain, while in Manta,nodules were dominated by the acid-sensitive strain.

4. Discussion

This experiment suggests a large effect of host genotype on thepattern of competition between strains; a large effect of host selec-tion. However, the competitive dominance of the acid-tolerant strainFB206 in the unamended acid soil in 3 of the 4 strain � inoculumcombinations indicates that it is possible to select strains for acid soilconditions that are also competitive; as has been demonstrated forR. tropici CIAT 899 and beans. Even so, the number of nodules formedand their size at low pH in both the control and the gypsum treat-ment for PI316937 were much smaller than for Manta. Thiscompetition at low pH may have been influenced by the relativerhizosphere population sizes, with the acid-tolerant strain havingmuch superior survival in acid soils than the acid-sensitive strain(Indrasumunar et al., 2011). However, it remains to be seen if an acid-tolerant strain can be selected that is competitive in a range of acidsoils and across a range of cultivars. Laboratory screening for acid-tolerance, and N2-fixation screening per se, does not address thishost selection component of inoculation success.

Although each seed received only 1 ml of inoculum in theplanting hole, the strains moved through the soil either by thewatering procedure or by growing with the root in the rhizosphere.Hence one of the aspects of inoculant selection that was notassessed in this experiment was the ability of strains to move withthe root tip and colonize the rhizosphere. This may be where theacid-sensitive strains gained their advantage at low pH in someinoculant combinations as the rhizosphere would not be as acidicas the bulk soil.

There was a difference in strain competition pattern betweensoybean cultivars in response to soil treatment. In PI416937, soiltreatment affected strain competitiveness in all 4 strain combinations(Fig. 2), but, in Manta, soil treatment only affected 1 strain combi-nation, FCB206 vs FCB146 (Fig. 2d). In PI416937, the acid-tolerantstrain FCB206 was superior to both acid-sensitive strains (FCB179and FCB146) in no added Ca treatment (Fig. 2c and d). Increasing Calevels in the gypsum treatment increased the competitive ability ofboth acid-sensitive strains resulting in them forming around 80% ofthe nodules tested.

Several studies have shown that Ca improves the ability of manyrhizobia to survive andpersist in acid soils, especially for strains that

Fig. 2. Effect of soil treatment on competition between acid-tolerant (FCB34, FCB206) and acid-sensitive (FCB179, FCB146) strains in two soybean cultivars (PI416937, Manta) grownin unamended acid soil (control), or soil treated with gypsum or lime. Values inside the histogram are the numbers of nodules tested.

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are acid-sensitive (Howieson et al., 1992; Ballen et al., 1998).Howieson et al. (1992) suggested that S. meliloti has a higherexternal Ca requirement at acid pH values, while Tiwari et al. (1992,1998) found that some acid-sensitivemutants of S.melilotiWSM419were Ca repairable and somewere not. In other studies, Ballen et al.(1998) found a much higher Ca requirement at low pH for the acid-sensitive Rhizobium etli than for R. tropici. Ballen et al. demonstratedamechanism bywhich low pH (pH 5.0) may affect properties of thecell envelope and suggested some roles of Ca (0.1,1.5 and 4.0mM) inminimizing these effects. Calcium concentration played a greaterrole inmaintaining cell envelope stability in the acid-sensitive R. etliUMR 1632 than in the acid-tolerant R. tropici strains, specifically insolute uptake and lipopolysaccharide structure, indicating thatacid-sensitive strains are more dependent upon Ca for resistingdamage caused by low pH. In the present experiment, increased Cain the gypsum treatmentminimized the acid stress effect on noduleformation by the acid-sensitive strains FCB179 and FCB146.

For cv. PI416937, the application of lime and gypsum thatincreased the soil pH as well as Ca concentration increased thecompetitiveness of the acid-sensitive strain FCB179 (Fig. 2c) so that itformed more than 95% of the nodules tested, but decreased thecompetitiveness of the other acid-sensitive strain FCB146 (Fig. 2b andd). Several studies of the effect of pH on strain competitiveness havealso demonstrated the competitive advantage of different acid-

tolerant strains in nodulating other plant species (Trifolium sub-terraneum, P. vulgaris, Medicago truncatula) at acid pH (Dughri andBottomley, 1983, 1984; Voss et al., 1984) with the more acid-tolerant strains adapted to colonise low pH soils (Robson andLoneragan, 1970). Thus, the better nodulation by the acid-sensitivestrain FCB179 with decreasing Hþ concentration is in agreementwith what has been reported elsewhere. In sterilized, limed andunlimed soil, Frey and Blum (1994) found that in the acidic soil (pH5.2) the acid-tolerant R. topici strain CIAT 899 out competed bothacid-sensitive strainsCIAT895andTAL182 in forming P. vulgarisbeannodules. This pattern of nodule occupancy changed in limed soil.When CIAT 899 was paired with TAL 182, the acid-sensitive straindominated bean nodulation, but, when CIAT 899 was paired withCIAT 895, both strains were equal in competitiveness. By contrast,Vargas and Graham (1989) found that the bean cultivar affected thecompetitive ability of the acid-tolerant strain CIAT 899 at pH 4.5, andthat at pH5.5 and 6.5 in sand culture the acid-sensitive strain becamethe most competitive, similar to our results.

The competitiveness of the acid-sensitive strain FCB146with bothacid-tolerant strains FCB34 and FCB206 on PI416937 nodulationwasinteresting (Fig. 2b and d).With no added Ca, the acid-sensitive strainFCB146 dominated the acid-tolerant strain FCB34, but was outcompeted by another acid-tolerant strain FCB206. In the gypsumtreatment, the acid-sensitive strain FCB146 dominated both acid-

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tolerant strains, but, in the lime treatment the acid-sensitive strainFCB146 was out competed by both acid-tolerant strains. It seemstherefore that the acid-sensitive strain FCB146 was very competitiveat low pH with high Ca (gypsum treatment) but less competitive athigher pH (lime treatment). This finding was not consistent with thefindings of Dughri and Bottomley (1983, 1984) for R. trifolii, and Vosset al. (1984) for Rhizobium phaseoliwho found that the acid-sensitivestrains were more competitive at high pH than in low pH.

Several studies showed that acid-tolerant and acid-sensitiverhizobia respond differentially to external Ca levels. Howieson et al.(1993) found that raising the Ca concentration from 1.0 to 10.0 mMincreased the attachment of both strains of R. meliloti to roots ofannual medics at pH 5.9. The attachment of the acid-sensitive strainCC169was increased7-foldwhilst that of the acid-tolerantWSM540was increased 4-fold. Raising the Ca concentration from 1.0 to10.0 mM at pH 6.6 increased the attachment of CC 169 nearly 3-fold,but not that ofWSM 540. The Ca effect on attachment was greater atpH 5.9 than at pH 6.6 for both strains of R. meliloti but the absolutelevel of attachment at pH 6.6 was greater than at pH 5.9.

In this study, the acid-tolerant strain FCB34, which was found tobe tolerant to acid stress in the acid agar media and in sterile acidsoils, was poorly competitive against both acid-sensitive strains onboth soybean cultivars (Fig. 2a and b). This strain was found to becompetitive only in the lime treatment, when paired with the acid-sensitive strain (FCB146) and inoculated on PI416937. Moreover,this strain was not able to form any nodules in any soil treatment onthe Manta cultivar. This suggests that competitive ability per se maybe the dominant factor in determining nodulation rather than pHtolerance.

Soybean cultivar Manta showed less variation in the pattern ofcompetition among soil treatments than for the PI416937. Therewas a host preference of Manta for the acid-sensitive strain FCB179which was stable over a range of different acid stress factors (Fig. 2aand c). The acid-sensitive strain FCB179 occupied all nodules testedwhen paired with the acid-tolerant strains FCB34 and FCB206 in allsoil treatments. Vargas and Graham (1988) similarly showed thatP. vulgaris cultivar had a strong influence on nodulation at acid pHwhere the selection was generally for the acid-tolerant strain.Montealegre et al. (1995) and Montealegre and Graham (1996)further identified a marked preference in the nodulation of beancultivar RAB39 for R. tropici UMR1899 (CIAT899) and demonstratedthe stability of this preference over different temperatures(17/12 �C, 24/19 �C, and 30/26 �C) and pH (4.5, 5.0, 5.5, and 6.5)conditions. They also found that the preference in nodulation ofRAB39 for R. tropici UMR1899 is effective against seven serologi-cally distinct strains of R. etli, the more commonly found strain insoils where the beans are grown.

Anyango et al. (1998) also found that the competitiveness ofR. tropici CIAT 899 vs R. leguminosarum bv. phaseoli strain Kim5 innodulating P. vulgaris was enhanced in a low pH 4.5 soil cf a pH 6.8soil (both previously sterilized by a irradiation) although it still onlyformed 30% of the nodules. Host plant cultivar intolerance of lowpH restricted N2-fixation. In our experiment, although the twocultivars used were relatively acid-tolerant increasing pH by limingto pH> 5 increased plant growth by about 50%. Both host and strainhave to be tolerant of the low pH in order for symbiosis to functioneffectively, and an acid-tolerant strain is required for survival of theinoculant in the bulk acid soil between crops of the legume host.These results and the present experiment suggest that the prefer-ence of a host cultivar for rhizobia strains that occur at lowfrequency in the majority of legume soils could be of majorsignificance in resolving strain competition problems in bean andsoybean, and particularly in acid soils.

Rhizobia strains nodulating soybean can be isolated from acidicand non-acidic soils where soybean has not been grown. In Brazil,

isolates from acidic soils were both fast growing (but differed fromSinorhizobium fredii) and slow growing (presumably bradyrhizo-bia). Twenty four percent of the strains isolated from undisturbedsoils were fast growing and 7 were acid-tolerant able to grow at pH4. Fast growing strains were also isolated from the cropped soilswhere soybean had been previously grown but in these soils rep-resented only 17% of the total soybean nodulating strains isolated.Most fast growing strains produced abundant polysaccharide(mucoid colonies) similar to strain FCB34 in our experiments(Hungria et al., 2001). Our other three strains had a similar colonyphenotype forming raised pinpoint colonies. Musiyiwa et al. (2005)isolated 129 slow growing “native” rhizobia with a range ofphenotypes from 92 Zimbabwe soils some with pH < 5. One isolatewas acid-tolerant to pH 3.5 and 16 to pH 4, and a further 82 to pH4.5, and all were able to nodulate either the promiscuous and/or thespecific soybean cultivars. An anomaly is that the acid-tolerantstrains were isolated from soils with pH < and > 5.

Our results showed that nodulation was more affected by soilpH than Ca level. Increasing the Ca level of the soil solution from1.9 mM to 13 mM only slightly increased nodule weight but notnodule number of soybean cv. Manta, but did not increase thenodulation of soybean cv. PI416937; while increasing soil pH from4.36 to 5.75 increased nodule number around 100% in both soybeancultivars and increased nodule fresh weight around 300% forPI416937 and 100% for Manta. On the other hand, strain competi-tiveness was more affected by Ca level than by soil pH. Increasingthe soil Ca level, but not pH, changed the competition pattern ofBradyrhizobium strains, where the competitiveness of the acid-sensitive strains increased resulting in them formingmore nodules.

This experiment shows how complicated is the selection ofrhizobia inoculants strains for use in acid soils. Merely selectingstrains from acid soils or for acid tolerance in vitro will not besufficient. Any presumptive inoculants strain will need to alsosurvive in acid soils which contain no plants to test survival in acidconditions per se as survival in the less acidic rhizosphere does notguarantee survival post plant death in soil. In our experiment hostcultivar had a dominating effect on both competition between anacid-tolerant and acid-sensitive strains in forming nodules andsubsequent nitrogen fixation. However the competitiveness of oneacid-sensitive strain with cv PI416937 was enhanced by increasingthe soil solution calcium concentration indicating that the calciummay be repairing cell damage resulting from low pH. Our resultsindicate that selection of inoculant strains for acid soils requiresassessment of their N2-fixation capacity per se at neutral pH, theirsurvival in acid soils, their affinity for cultivar(s) being grown (hostselection factor in strain selection). These factors can only betested by inoculant trials in non sterile acid soils and this requiresa marker to identify whether the inoculant strain has formed thenodules. Such field trials need to be conducted over severalseasons and soil types to assess the strain stability in differentenvironmental conditions. The amount of lime required to costeffectively boost nodulation and N2-fixation is another variable tobe addressed as soil pH per se in our experiment had a major effecton N2-fixation by soybean. There is no quick fix for overcomingthis intractable problem of poor nodulation and N2-fixation inacid soils.

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