Moderately acidophilic mesorhizobia isolated ... - Wiley Online Library

Letters in Applied Microbiology ISSN 0266-8254

ORIGINAL ARTICLE

Moderately acidophilic mesorhizobia isolated from chickpea C. Brı´gido1, A. Alexandre1,2, M. Laranjo1,2 and S. Oliveira1,2 1 Laborato´rio de Microbiologia do Solo – ICAM (Instituto de Cieˆncias Agra´rias Mediterraˆnicas), Universidade de E´vora, Portugal 2 Departamento de Biologia, Universidade de E´vora, Portugal

Keywords chickpea (Cicer arietinum L.), moderately acidophilic, pH, rhizobia, symbiotic effectiveness. Correspondence Solange Oliveira, Departamento de Biologia, Universidade de E´vora, Apartado 94, 7002-554 E´vora, Portugal. E-mail: [email protected]

2006/0709: received 18 May 2006, revised 2 August 2006 and accepted 19 September 2006 doi:10.1111/j.1472-765X.2006.02061.x

Abstract Aims: Our aim was to evaluate the effect of acid and alkaline pH on chickpea rhizobia, and on chickpea–rhizobia symbiosis. Methods and Results: Forty-seven rhizobia isolates obtained from 12 Portuguese soils were grown at pH 5, 7 and 9. Among these, 26 grew more at pH 5 than at 7, suggesting the existence of acidophiles. All isolates were identified as mesorhizobia by 16S rDNA partial sequence analysis. Molecular phylogeny of isolates based on partial 16S rDNA sequences suggests that pH tolerance might be species related. Further studies were conducted with six isolates, which were able to grow at acid pH. Isolates PT-35 and 64b grow optimally at pH 6–5–7, with a minimal pH range from 5 to 3, and may thus be considered as moderately acidophilic. Both isolates belong to a previously identified putative new Mesorhizobium species, based on 16S rDNA sequence. Conclusions: Two moderately acidophilic mesorhizobia isolated from chickpea were identified (PT-35 and 64b). A positive correlation was found between the symbiotic effectiveness at low pH and the acid tolerance of rhizobia isolates. Significance and Impact of the Study: This is the first report on moderately acidophilic mesorhizobia, and is an important contribution for the development of highly effective inoculants for chickpea in acid soils.

Introduction Legumes have the almost unique advantage of being able to establish nitrogen-fixing symbioses with bacterial microsymbionts (rhizobia). This association delivers nitrogen to the host plant without the need for chemical fertilizers, and provides a nitrogen supplement for the crops to be cultivated afterwards (Priefer et al. 2001). Severe environmental conditions are limiting factors for the growth and activity of both nitrogen-fixing partners (Zahran 1999). Several authors (Singleton et al. 1982; Priefer et al. 2001) have suggested that nodule formation and nitrogen fixation are highly affected by stress conditions. Acid pH limits the persistence of rhizobia strains in the soil (Graham 1992), affecting the exchange of molecular signals between rhizobia and their hosts, thus reducing nodulation (Graham et al. 1994; Hungria and Vargas 2000). Nevertheless, legumes and their rhizobia exhibit varied responses to acidity. For example, chickpea (Cicer arietinum L.) is a successful legume on alkaline soils (Rao 168

et al. 2002), while the symbiosis with rhizobia is better adapted to acidity (Siddique et al. 1999; Howieson et al. 2000). Somasegaran and Hoben (1994) described optimal pH range for rhizobia as 6–7, and Jordan (1984) reported the tolerance pH range in strains of Rhizobiaceae to be 4Æ5–9Æ5. The ability of soil bacteria from the family Rhizobiaceae to survive in acid media and soils has been assessed, and some acid-tolerant strains have been identified (Graham 1992; Glenn and Dilworth 1994; Graham et al. 1994). Different species of rhizobia vary in their response to low pH. Strains of Mesorhizobium ciceri, which nodulate chickpea, tolerate pH values from 5Æ0 to 10Æ0 (Nour et al. 1994). However, another chickpea symbiont, Mesorhizobium mediterraneum, was described as acid sensitive, with a pH range of 7–9Æ5 (Nour et al. 1995). Previous studies on the effect of heat and pH stress on chickpea rhizobia revealed acid-tolerant isolates (Rodrigues et al. 2006). The aims of this study were to determine the tolerance of Portuguese chickpea rhizobia isolates to different pH

ª 2006 The Authors Journal compilation ª 2006 The Society for Applied Microbiology, Letters in Applied Microbiology 44 (2007) 168–174

C. Brı´gido et al.

values, and to evaluate their symbiotic performance. We investigated 47 chickpea rhizobia isolates, collected from 12 different Portuguese soils, which were identified by 16S rDNA partial sequence analysis. This study may contribute to the selection of new rhizobial strains adapted to low pH ecosystems to be used as chickpea inoculants. Materials and methods Isolation of rhizobia Isolates were obtained from root nodules of chickpea trap plants grown in pots, using different soils from diverse Portuguese regions (Aveiro, Leiria-Caldas da Rainha, Castelo Branco, Coimbra, Portalegre, Santare´m, Castelo Branco-Telhado and Viseu), as described by Somasegaran and Hoben (1994). Isolates from Beja, E´vora, Elvas and Setu´bal were previously obtained (Laranjo et al. 2001, 2002; Rodrigues et al. 2006). Isolates were maintained in tryptone yeast (TY) agar slants at 4C, and kept in 30% glycerol at )80C.

Moderately acidophilic mesorhizobia from chickpea

conditions were pH 7 and 28C. The isolates were grown in liquid medium at 28C, during 48 h. Three replicates per treatment were done. Growth quantification was recorded as optical density (OD) at 540 nm. An inoculum was used so that the initial OD was 0Æ03. For some isolates (29-Beja, CR-32, 64b-Beja, 75-Elvas, C-1 and PT-35), growth in medium at pH 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12 was also quantified. The liquid medium at pH 1–5 was buffered with acetate buffer, while at pH 6 and 7, it was buffered with MES and for alkaline liquid media (pH 8–12), AMPD was used as buffer. Symbiotic performance at different pH values

The 16S rRNA gene was amplified by PCR, using primers Y1 (Young et al. 1991) and Y3 (Laranjo et al. 2004). The PCR products were purified using GFXTM PCR DNA and Gel Band Purification kit (GE Healthcare, Buckinghamshire, UK) following the manufacturer’s instructions. Two primers, IntF and IntR, were used as internal primers for double-stranded sequencing (Laranjo et al. 2004). The sequences were analysed and aligned using BioEdit (Hall 1999). Molecular phylogeny was reconstructed using MEGA3 (Kumar et al. 2004), and a dendrogram was generated by the Neighbour-Joining method. The 16S rDNA sequences obtained from native isolates were compared with those of rhizobial reference strains available in the GenBank database.

The symbiotic performance of rhizobia isolates was determined under different pH conditions, by inoculation of chickpea plants, as described previously (Somasegaran and Hoben 1994). Chickpea plants (cultivar Chk 3226) were grown in plastic pots filled with sterile vermiculite in the growth chamber. A nitrogen-free nutrient solution was used (Laranjo et al. 2002). Uninoculated nitrogenfree plants served as negative controls, whereas uninoculated nitrogen-supplemented plants were used as positive controls. To study the symbiotic performance under pH stress, the pH of the nutrient solution was adjusted to 5 or 7. Three replicates were used for each treatment, and the plants were harvested 8 weeks after sowing. Parameters measured were shoot dry weight (SDW), root dry weight (RDW), number of nodules (NN) and nodules dry weight (NDW). Symbiotic effectiveness (SE) was calculated according to Laranjo (2002). In order to compare the symbiotic effectiveness and other relevant parameters of different isolates under different pH values, the positive control SDW was considered as 100%. Data represent average of three replicates per treatment. One-way anova and Duncan’s multiplerange test were carried out using SPSS 13.0 (SPSS, Chicago, IL, USA). Spearman correlations were performed.

Plasmid profiles

Results

Plasmid profile analysis was performed as described by Laranjo et al. (2001).

Strain isolation

Survival of rhizobial isolates at various pH values

Forty-seven isolates were obtained from 12 soils from different Portuguese regions (Table 1). The ability of these isolates to nodulate chickpea was confirmed by re-inoculation on chickpea plants.

16s rDNA sequencing

To evaluate the tolerance to alkaline conditions (pH 9), yeast extract mannitol (YEM) medium was buffered with 26-mmol l)1 2-amino-2-methyl-1,3-propandiol (AMPD). For pH 5, YEM was buffered with 25-mmol l)1 acetate buffer. To adjust the medium to pH 7, 20-mmol l)1 2-morpholinoethanesulfonic acid (MES) was added, as described by Priefer et al. (2001). The control growth

Strain identification by 16S rDNA sequencing The 47 isolates used in this study were found to belong to the Mesorhizobium genus by 16S rDNA partial sequence analysis (data not shown).


169



Table 1 Origin and soil pH, total number of isolates and number of isolates with maximum growth at different pH values (5, 7 and 9)

1

2

3

4

5

Number of isolates with maximum growth

Origin

Number of isolates

Soil pH

pH 5

pH 7

pH 9

Aveiro Beja Caldas da Rainha Coimbra Castelo Branco Elvas E´vora Portalegre Santare´m Setu´bal Telhado Viseu

2 5 5 7 6 4 5 1 1 5 1 5

5Æ5 8Æ2 6Æ8 5Æ7 6Æ5 5Æ8 5Æ9 5Æ3 7Æ8 8Æ1 7Æ3 5Æ9

0 2 0 7 4 4 4 1 0 0 0 4

2 3 5 0 2 0 1 0 1 5 1 1

0 0 0 0 0 0 0 0 0 0 0 0

Figure 1 Example of plasmid profiles gel. Lane 1, 29-Beja; lane 2, A-6-Aveiro; lane 3, STR-16-Santare´m; lane 4, 78-Elvas; lane 5, 85-Elvas.

Plasmid profiles Evaluation of rhizobial isolate tolerance to various pH values (5, 7 and 9) The growth of the 47 rhizobial isolates was evaluated under pH 5, 7 and 9 (Table 1). Interestingly, 26 isolates showed maximum growth at pH 5, suggesting the presence of moderately acidophilic native strains. At pH 9, 32 isolates showed growth inhibition, suggesting that they were alkaline-sensitive. Twenty-one isolates showed optimal growth at pH 7, suggesting that they were neutrophiles. No isolate showed maximum growth at pH 9. A positive correlation (r ¼ 0Æ5; P < 0Æ01) was depicted between maximum growth pH and the origin-soil pH of isolates. Isolates from acidic or neutral soils revealed higher growth at pH 5 or 7, and isolates from alkaline soils showed a preference for neutral pH. From the 47 isolates, six, able to grow at acid pH, were selected for further studies. Two of them showed maximum growth at pH 7 (29 and CR-32), and the other four showed maximum growth at pH 5 (75, 64b, PT-35 and C-1).

Plasmid profiles of the 47 isolates were consistent and reproducible. Figure 1 is an example of an agarose gel showing plasmid profiles. The isolates revealed plasmid number ranging from 0 to 6 (data not shown). In most isolates, one or two large plasmids were detected. A negative correlation (r ¼ )0Æ435; P < 0Æ01) was found between the plasmid number and maximum growth pH of the isolates. Four out of five (80%) isolates with a higher plasmid number showed preference for pH 5, while 15 out of 20 (75%) isolates without plasmids grew better at pH 7. Table 2 presents the plasmid number and the plasmid molecular weight estimated for the six selected isolates. Evaluation of isolate growth at pH 1–12 The growth of the six selected isolates was further evaluated at pH 1–12 (Fig. 2). Despite all being able to grow at acid pH, these isolates revealed different optimal growth pH and different tolerance pH range (Table 2). Isolate CR-32 grew optimally at pH 7–6–5, while 29

Table 2 Rhizobial isolates, origin, number of plasmids and survival range pH values Origin

Isolate

Plasmid number

Plasmids molecular weight (kb)*

Optimal growth pH

Tolerance pH range

Coimbra Portalegre Beja

C-1 PT-35 29 64b 75 CR-32

1 1 6 1 1 0

240 230 15/20/140/145/210/260 440 210

6–8–5 6–5–7 7–6–8 6–5–7 6–5–8 7–6–5

4–9 3–9 5–8 3–9 3–9 4–9

Elvas Caldas da Rainha

*29-Beja isolate was used as standard for the determination of plasmid molecular weights. Reported by Laranjo et al. (2001).

170




2·00 1·80 1·60 1·40

OD

1·20 1·00 0·80 0·60 0·40 0·20 0·00 CR-32

29

64b·

PT-35

C-1

75

Figure 2 Growth of chickpea rhizobia from five locations of Portugal at 12 different pH values: Caldas da Rainha (CR-32), Beja (29 and 64b), Portalegre (PT-35), Coimbra (C-1) and Elvas (75). pH 1; pH 2; pH 3; pH 4; pH 5; pH 6; pH 7; pH 8; pH 9; pH 10; pH 11; pH 12.

isolate showed a higher growth at pH 7–6–8, indicating that they are neutrophiles. However, these isolates revealed different tolerance pH ranges. Isolate CR-32 showed tolerance from pH 4 to 9, while isolate 29 grew from pH 5 to 8. Isolates 64b and PT-35 grew optimally at pH 6–5–7, with a minimal pH for growth of 5–3, showing that these isolates are moderately acidophilic. Both isolates showed a pH range of 3–9. The isolates C-1 and 75 grew better at pH 6–8–5 and 6–5–8, respectively. The pH range for the isolate C-1 was 4–9 and for the isolate 75 it was 3–9. Phylogeny based on 16S rDNA sequencing According to the complete 16S rDNA sequence-based phylogeny, isolates PT-35, C-1, 75, CR-32, 29 and 64b were distributed by four main clusters (Fig. 3). Isolates from Portalegre, Beja and Coimbra on cluster A form an independent branch apart from the closest type strains, supporting the new species group suggested by Laranjo et al. (2004). The sequence identity of the 16S rDNA sequence of these isolates ranged from 99Æ7% to 99Æ9%. On cluster B, isolate CR-32 groups with Mesorhizobium tianshanense (99Æ9%). Isolates close to M. tianshanense that nodule chickpea have already been reported (Laranjo et al. 2004). Isolate 29 groups with Mesorhizobium temperatum and M. mediterraneum, corresponding to the cluster C (Laranjo et al. 2004). Isolate 75 belongs to the cluster D, and is closer to Mesorhizobium loti (99Æ8%) (Laranjo et al. 2004).

Symbiotic effectiveness at different pH values The symbiotic effectiveness (SE) of rhizobia isolates 29 (neutrophile), C-1 (similar growth at pH 5 and 7) and 64b (moderate acidophile) was evaluated under pH 5 and 7 (Table 3). The SE values obtained ranged from 21Æ4% to 59Æ8%. The number and dry weight of nodules were also analysed. No correlation was found between the number of nodules and SE. For each strain, all parameters analysed (except one case, nodule number in C-1) were higher at the pH value which was preferred for the isolate growth in liquid media (pH 7 for 29 and pH 5 for 64b and C-1). The moderately acidophilic isolate (64b) showed a higher SE value under pH 5, and the neutrophile isolate (29) demonstrated a higher symbiotic effectiveness at pH 7. These results indicate that the symbiotic performance of the isolates is significantly dependent on soil pH, probably because it affects bacterial growth and persistence. Discussion Chickpea is the third most widely grown grain legume in the world, and constitutes an important source of protein. A significant constrain to chickpea crops is the pH stress, which limits the nodulation and the nitrogen fixation (Graham 1992); hence, rhizobia strains tolerant to pH stress may improve the legume crop production. Acid and alkaline sensitivity of micro-organisms can be defined experimentally as the inability to grow on


171



94 M. amorphae 56 M. septentrionale 51 M. plurifarium 91 M. huakuii PT-35-Portalegre (a)

88 64b-Beja 70 87 C-1-Coimbra 97 M. tianshanense

(b)

CR-32-Caldas da Rainha 38

M. temperatum

75 100

100

M. mediterraneum

(c)

29-Beja M. chacoense M. ciceri 100 M. loti 75 75-Elvas

100

(d) R. etli

100

R. leguminosarum 76

S. medicae 100

Figure 3 Neighbour-Joining phylogeny of chickpea rhizobia based on 16S rDNA sequences (alignment length 1353 bp). Percentage bootstrap support (1000 replicates) is indicated on internal branches; scale bar indicates 0Æ01 substitutions per site.

S. meliloti

A. caulinodans B. japonicum 0·01

Table 3 Shoot dry weight (SDW), root dry weight (RDW), number of nodules (NN), nodule dry weight (NDW) (mean value of three replicates ± SD) and symbiotic effectiveness (SE) of three isolates (29-Beja, 64b-Beja and C-1-Coimbra) at pH 5 and 7 Isolate

pH

SDW (g)

29-Beja

5 7 5 7 5 7

0Æ577 0Æ664 1Æ610 1Æ384 1Æ221 0Æ809

64b-Beja C-1-Coimbra

± ± ± ± ± ±

RDW (g) 0Æ171 0Æ154 0Æ039 0Æ159 0Æ366 0Æ188

0Æ414 0Æ530 0Æ607 0Æ530 0Æ643 0Æ490

± ± ± ± ± ±

NN 0Æ039 0Æ048 0Æ030 0Æ029 0Æ109 0Æ061

41 53 106 69 133 156

± ± ± ± ± ±

12 11 13 12 11 45

NDW (g)

SE* (%)

0Æ178 0Æ332 0Æ198 0Æ156 0Æ225 0Æ183

21a 24a 60c 51bc 45b 30a

± ± ± ± ± ±

0Æ045 0Æ162 0Æ011 0Æ016 0Æ071 0Æ066

*SE: SDW inoculated plants/SDW noninoculated control plants. In the SE column, means denoted with different letters differ significantly (P ¼ 0.05) based on Duncan’s multiple range test.

minimal medium buffered to pH 5 or 9, respectively (Riccillo et al. 2000). The tolerance to low pH has been previously observed in rhizobial isolates (Jordan 1984; Martıńez-Romero et al. 1991; Graham 1992), namely in chickpea mesorhizobia (Nour et al. 1994; Rodrigues et al. 2006). Maaˆtallah et al. (2002) found that chickpea-infective strains are more tolerant than rhizobia in general, with a pH range of 5–8. However, despite all the reports on acid-tolerant strains, this is the first description, to our knowledge, on Mesorhizobium sp. isolates that may be 172

considered as moderately acidophilic owing to their optimal growth at pH 6–5–7 and tolerance range at pH 3–9. According to the molecular phylogeny based on 16S rDNA partial sequence analysis (data not shown), additional isolates that belong to the group of Mesorhizobium amorphae/Mesorhizobium plurifarium/Mesorhizobium septentrionale showed a preference for pH 7, while the isolates which belong to the hypothetical new species, already reported by Laranjo et al. (2004), revealed a maximum growth at pH 5. The isolates grouping with



M. temperatum/M. mediterraneum and the ones from M. tianshanense cluster, grow better at pH 7. The isolates that belong to the group of M. ciceri/M. loti showed a preference for both pH 5 and 7. These results suggest a species-related tolerance to acidity, and are in disagreement with Amarger et al. (1997), who suggested that tolerance to acidity is more strain specific than species specific. Our results revealed a positive correlation (r ¼ 0Æ580, P < 0Æ001) between isolate species and origin soil pH, agreeing with Martıńez-Romero et al. (1991), who suggest that soil pH seems to select the species that are successful for legume nodulation. A positive correlation was found between the maximum growth pH of the isolates and their origin soil pH, agreeing with some previous reports (Surange et al. 1997; Kulkarni and Nautiyal 1999; Rodrigues et al. 2006). These results may suggest an easier adaptation of rhizobia to acidic environmental conditions than to alkaline ones. However, Priefer et al. (2001) detected no correlation between bean rhizobia tolerance to different pH values and the origin-soil pH. Plasmid profile analysis of the isolates revealed diversity in plasmid number and molecular weight. Several isolates from the same geographic area showed identical plasmid profiles. These results suggest genetic differences among rhizobial populations of the different soil areas, already described by Laranjo et al. (2001, 2002). A negative correlation was found between plasmid number and maximum growth pH of the isolates, supporting the suggestion of Harrison et al. (1989), who proposed that these genetic differences on Rhizobium populations could be related to soil pH, although no such relationship was found by Thurman et al. (1985). The effect of pH on nodulation and symbiotic effectiveness was investigated in three strains, including one moderately acidophilic. We found a direct relationship between optimal growth pH and the pH for higher symbiotic effectiveness of the isolate. This relationship between the symbiotic effectiveness at low pH and the acid tolerance found in rhizobia nodulating chickpea was also reported in peanut rhizobia, by Angelini et al. (2005). It is possible that symbiotic performance of the isolates is significantly dependent on soil pH, because it affects, at least, bacterial growth. This suggests that the achievement of a high symbiotic performance of rhizobia inoculants in chickpea crops should require the selection of a strain whose preferred pH is similar to the pH of the soil to be cultivated. However, studies by Içgen et al. (2002) showed that chickpea rhizobia strains displayed a tendency to neutralize the pH of the medium when grown freely in media adjusted to different pH values, and this might account for the success in the establishment of symbiosis in both acidic and alkaline conditions. The present study showed that acid pH leads to a


decrease in the number of nodules formed in plants inoculated with an acid-sensitive isolate, agreeing with other studies (Angelini et al. 2005; Chemining’wa and Vessey 2006). Previous studies have indicated a correlation between strain performance under stress in pure culture and strain behaviour under symbiotic conditions, namely for temperature stress (Kulkarni and Nautiyal 1999). Similarly, the present work shows that in vitro evaluation of strain growth under pH stress may also be a useful method for finding rhizobial isolates adapted to different soil pH. Here we report two moderately acidophilic chickpea mesorhizobia that could potentially be used for preparing highly effective inoculants for chickpea plants growing in acid soils. The present study reinforces that potential inoculants should be selected taking into account both soil pH and maximum growth pH of the strain. To confirm these results in the field, further research is required to elucidate the competitiveness of these isolates, when compared with the effective population of chickpea rhizobia in the soil. Acknowledgements This work was supported by Fundaçaõ para a Cieˆncia e Tecnologia (FCT), and co-financed by EU-FEDER (POCTI/BME/44140/2002). A. Alexandre (SFRH/BD/ 18162/2004) and M. Laranjo (SFRH/BD/6772/2001) acknowledge the Ph.D. fellowships from FCT. The authors thank G. Mariano for her technical assistance. References Amarger, N., Macheret, V. and Laguerre, G. (1997) Rhizobium gallicum sp. nov. and Rhizobium giardinii sp. nov., from Phaseolus vulgaris nodules. Int J Syst Bacteriol 47, 996–1006. Angelini, J., Taurian, T., Morgante, C., Ibanez, F., Castro, S. and Fabra, A. (2005) Peanut nodulation kinetics in response to low pH. Plant Physiol Biochem 43, 754–759. Chemining’wa, G.N. and Vessey, J.K. (2006) The abundance and efficacy of Rhizobium leguminosarum bv. viciae in cultivated soils of the eastern Canadian prairie. Soil Biol Biochem 38, 294–302. Glenn, A.R. and Dilworth, M.J. (1994) The life of root nodule bacteria in the acidic underground. FEMS Microbiol Lett 123, 1–9. Graham, P.H. (1992) Stress tolerance in Rhizobium and Bradyrhizobium, and nodulation under adverse soil-conditions. Can J Microbiol 38, 475–484. Graham, P.H., Draeger, K.J., Ferrey, M.L., Conroy, M.J., Hammer, B.E., Martinez, E., Aarons, S.R. and Quinto, C. (1994) Acid pH tolerance in strains of Rhizobium and


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