Nodulation and growth of common bean (Phaseolus

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Soil Biology & Biochemistry 39 (2007) 1744–1750 www.elsevier.com/locate/soilbio

Nodulation and growth of common bean (Phaseolus vulgaris) under water deficiency Bacem Mnasri, Mohamed Elarbi Aouani, Ridha Mhamdi Laboratoire Interactions Le´gumineuses-Microorganismes, Centre de Biotechnologie, Technopole de Borj-Ce´dria, BP 901, Hammam-Lif, Tunisia Received 16 November 2006; received in revised form 15 January 2007; accepted 23 January 2007 Available online 5 March 2007

Abstract Three experiments were conducted in order to investigate the effect of water deficiency on nodulation, rhizobial diversity and growth of common bean. In the first experiment, the effect of water deficiency was studied on two soil samples under glasshouse conditions. A significant decrease in nodulation and shoot dry weight production was observed. The molecular characterization of the root nodule isolates by PCR-RFLP of 16S rRNA and nodC genes showed that the nodulation by Rhizobium etli was severely inhibited. The in vitro analysis of salt tolerance indicated that drought stress favoured nodulation by salt-tolerant strains. In the second experiment, the effect of water deficiency was studied on sterilized sand using Rhizobium tropici CIAT899T and Ensifer meliloti bv. mediterranense 4H41 as inoculants. The results showed that strain 4H41, which is the more salt tolerant, was more competitive and more effective under water deficiency than strain CIAT899T. In the third experiment, the strain 4H41 was used to inoculate four fields. A significant increase in nodule number, shoot dry weight and grain yield was observed even in the non-irrigated soils. This work constitutes the first report of a strain enhancing the growth and the grain yield of common bean under water deficiency. r 2007 Elsevier Ltd. All rights reserved. Keywords: Drought stress; Ensifer meliloti; Field inoculation; Genetic diversity; Nitrogen fixation; Salt tolerance

1. Introduction Common bean (Phaseolus vulgaris) is considered a poor nitrogen-fixer pulse in comparison to other grain legumes (Hardarson, 1993). Sparse nodulation and the lack of response to inoculation in field experiments had been frequently reported worldwide, raising questions about the benefits of inoculation (Graham, 1981; Buttery et al., 1987). This fact is attributed to intrinsic characteristics of the host plant, particularly the nodulation promiscuity (Michielis et al., 1998), as well as the great sensitivity to other nodulation-limiting factors, such as the high rate of N-fertilizer used in intensive agriculture, nutrient deficiency, high temperatures and soil dryness (Graham, 1981). Water deficiency is a major limiting factor of plant productivity and symbiotic nitrogen fixation in many arid regions of the Mediterranean basin. One of the immediate responses of rhizobia to water deficiency concerns the Corresponding author. Tel.: +21 671 430 855; fax: +21 671 430 934.

E-mail address: [email protected] (R. Mhamdi). 0038-0717/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2007.01.030

morphological changes (Shoushtari and Pepper, 1985; Busse and Bottomley, 1989). The modification of rhizobial cells by water stress will eventually lead to a reduction in infection and nodulation of legumes (Hunt et al., 1981; Zahran and Sprent, 1986). In addition to its depressive effect on nodule initiation, water deficit also results in restriction of nodule development and function (Serraj et al., 1999). The occurrence of rhizobial populations in desert soils and the effective nodulation of legumes growing therein (Jenkins et al., 1989; Waldon et al., 1989) emphasize the fact that rhizobia can exist in soils with limiting moisture levels; however, population densities tend to be lowest under the most desiccated conditions and to increase as the moisture stress is relieved. It is well known that some free-living rhizobia are capable of survival under drought stress or low water potential (Fuhrmann et al., 1986). The wide range of moisture levels characteristic of ecosystems where legumes have been shown to fix nitrogen suggests that rhizobial strains with different sensitivity to soil moisture can be selected. Laboratory studies have shown that sensitivity to moisture stress varies for a variety

ARTICLE IN PRESS B. Mnasri et al. / Soil Biology & Biochemistry 39 (2007) 1744–1750

of rhizobial strains (Fuhrmann et al., 1986; Busse and Bottomley, 1989; Osa-Afina and Alexander, 1982). It has been reported that nodulation and nitrogen fixation in alfalfa can be improved by inoculating plants with competitive and drought tolerant rhizobia (Zahran, 1999). Thus, we can reasonably assume that rhizobial strains can be selected with moisture stress tolerance within the range of their legume host which is generally more sensitive to moisture stress than bacteria (Zahran, 1999). A research program was launched in the frame of the project Aquahiz. The primary goal of this project is to increase the production of chickpea, common bean and faba bean in Algeria, Egypt, Morocco and Tunisia, where their nutrition and yield are affected by water deficit. The project is founded on the preceding evidence for inter and intraspecific genetic variability of legumes for tolerance to water deficit and in the demonstrated capacity of specific rhizobia to enhance the tolerance to water deficit by the plant. This project was designed to develop the scientific and technical bases and knowledge required to determine when, where, and how management of symbiotic nitrogen fixation may be an appropriate and effective way to stabilize and increase benefit in the legume-based cropping systems. The immediate objective of the work presented in this paper is to study the effect of induced water deficiency on nodulation, rhizobial diversity and growth of common bean. 2. Material and methods 2.1. Water-deficiency experiment under soil conditions Two soil samples originating from the north of Tunisia, A (Ben Mustapha) and B (Maghraoui) were used to study the effect of water deficiency on nodulation and diversity of nodulating rhizobia under glasshouse conditions. The two sites belonged to the sub-humid stage and had been cultivated with cereals in rotation with three main grain legumes, faba bean, chickpea and common bean. The soils A and B were non-saline and slightly alkaline, A is sandy clay and B is clay. The soil samples were distributed in 3 l plastic pots (2 kg per pot) and transferred to the glasshouse. The seeds of common bean cv. Flamingo were surface sterilized by immersion for 10 min in 0.2% HgCl2 and extensively washed with sterile distilled water. Two seeds were sown per each pot. Two treatments were considered, a control treatment which received 150 ml of water three times per week and a water-deficient treatment which received 150 ml one time per week. The irrigation dose of 150 ml corresponds to field capacities of 37% and 25% for soils A and B, respectively. The plants were harvested after 2 months. Shoots were oven dried at 70 1C for 48 h and weighted. Nodules were counted for each plant and stored in small vials containing dehydrating powder (CaCl2) for later rhizobia isolation and characterization. Ten individual plants were considered for each treatment. Statistical analysis was done using the STATISTICA

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software by the ANOVA/MANOVA program. The HSD test (Po0.05) was used for means comparison. Rhizobia were isolated from the root nodules according to the standard procedures as previously described (Mhamdi et al., 1999). One isolate was kept from each nodule. Nodules produced on the water-deficient plants were all used. Nineteen nodules were recovered from the control plants for each soil sample. PCR-RFLP of the 16S rRNA genes was conducted on cells treated with proteinase-K using primers fD1 and rD1 (Weisburg et al., 1991) as previously described (Mhamdi et al., 2002). Species assignation was done according to 16S rDNA typing using MspI and NdeII and compared with the published database of mapped restriction sites in the 16S rRNA genes of rhizobia (Laguerre et al., 1997). The NodC genes were amplified using primers nodCi and nodCf and digested with MspI as previously described (Mhamdi et al., 2002). Salt tolerance of the different isolates was assessed in yeast-extract mannitol (YEM) broth supplemented with different concentrations of sodium chloride going from 0.2% to 2% (w/v). Rhizobia were taken from cultures stored in 20% glycerol at 80 1C and transferred to YEM slants. Transfers were made from the slants to YEM broth and inoculated flasks were incubated on a rotary shaker at 150 rev min1 and 28 1C. At mid-exponential growth phase, the equivalent of 20 ml of broth culture for OD620 nm ¼ 1 was transferred to 30 ml flasks containing 5 ml of YEM broth with varying concentrations of NaCl. Three replicates were considered for each test. The ability to grow was checked visually after 5 days of incubation at 28 1C under orbital agitation. 2.2. Water-deficiency experiment on sterilized sand Plastic pots (500 ml) filled with sterilized sand were used as growth support. The sand was extensively washed with water before autoclaving. One pre-sterilized and germinated seed was grown in each pot. Both cultivars Coco and Flamingo were used. The pots were covered with lids to ensure maximum sterility. After 3 days, the seedlings were inoculated and the cotyledons were kept out through a hole managed on the lid. Two rhizobial strains, Rhizobium tropici CIAT899T and Ensifer meliloti 4H41 (Mnasri et al., 2006) were used for inoculation. Four inoculation treatments were performed: non-inoculated, inoculated with CIAT899T (106 cfu), inoculated with 4H41 (106 cfu) and coinoculated with CIAT899 and 4H41 (1/2:1/2). The plants were subdivided into two sets. The first set of control plants was kept to 90% of field capacity (FC) with a sterilized nitrogen-free solution (Vadez et al., 1996). The second set of water-deficient plants was maintained at 30% FC. Regular weightings were performed every two days to restore the moisture levels. Thirty-five days after inoculation, the plants were harvested and subjected to analyses of nodule number and shoot dry yield. Ten individual plants were considered for each treatment. The statistical analyses were performed as previously stated.

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The nodules from the co-inoculated plants were collected and used for nodule-occupancy monitoring. The rhizobia were isolated as previously stated and one isolate was kept from each single nodule. A total of 150 isolates (15 10 plants) were used for each cultivar (cv. Coco and cv. Flamingo) and each irrigation regime (90% FC and 30% FC). The nodule occupancy by strains 4H41 and CIAT899T was determined according to the ability to grow in YEM broth supplemented with 3% NaCl as stated above. The maximum salt tolerance of strain CIAT899T in YEM broth is about 1.8% NaCl; however, strain 4H41 supported the growth in salt concentrations higher than 4% NaCl (Mnasri et al., 2006). 2.3. Field inoculation experiments The on-farm trials were conducted in close partnership with farmers and extension services. They were replicated across farmers’ fields but not within fields. Four fields were selected, A (Ben Mustapha), C (Lazhar), D (Tej) and E (El Kef). A, C and D belonged to the sub-humid stage and E to the semi-arid stage. The field A received a supplement of irrigation according to plant need while the other fields were not irrigated. The experimental design consisted of two treatments, not inoculated and inoculated. Each treatment was subdivided into two subplots cultivated with cultivars Coco and Flamingo, respectively. The subplots were 13.5 6 m in size and contained 30 rows. The sowing density was 20 seeds/m2 (10/50 cm). A margin of 2 m separated the inoculated and the non-inoculated plots. The experiment was established in March 2006. The native strain E. meliloti bv. mediterranense 4H41 (Mnasri et al., 2006) was grown to late exponential phase in YEM (Vincent, 1970) and diluted to 1/20 with well water and then used to inoculate the emerging seedlings using a watering can (approximately 5 ml for each). At flowering stage, 30 plants were randomly collected from each subplot and used for nodule counting and shoot dry weight analysis. At final harvest, 30 plants were also used for grain yield determination. Results were subjected to analysis of variance. 3. Results 3.1. Effect of water deficiency on nodule number and shoot dry weight Water deficiency induced a significant decrease in nodulation and shoot dry weight of common bean cultivated on both soil samples used comparing to the control plants (Table 1). 3.2. Effect of water deficiency on the genetic diversity of nodulating rhizobia The rhizobia nodulating common bean under both regimes of irrigation were isolated from the root nodules.

Table 1 Effect of water deficiency on nodule number and shoot dry weight of common bean cultivated on soil samples in the glasshouse Soil

A B

Nodule number (per plant)

Shoot dry weight (g/plant)

Control

Water deficient

Control

Water deficient

10 29

1.5 1

1.07 1.06

0.42 0.30

Values are means of 10 replicates. The difference between the control and the water-stressed plants is significant (po0.05).

Table 2 Diversity of the rhizobia nodulating common bean in the glasshouse under control and water-deficient conditions Soil

Species

nodC type

Number of isolates Control

Water deficient

A

Rhizobium gallicum

a

19/19

4/4

B

Rhizobium etli Sinorhizobium fredii Rhizobium gallicum Agrobacterium sp.

c d a —

10/19 8/19 0/19 1/19

0/6 2/6 4/6 0/6

Few isolates were obtained from the water-stressed plants because of the low number and size of nodules (data not shown). The typing by PCR-RFLP of 16S rRNA and nodC genes showed that water deficiency severely inhibited the nodulation by R. etli compared to other species (Table 2). 3.3. Salt tolerance of isolates The different isolates were tested for their capacity to grow in broth medium supplemented with increasing concentrations of salt (Table 3). Our results showed that the application of water stress favoured the nodulation by salt-tolerant rhizobia. All the isolates recovered under water deficiency tolerated at least 1.2% NaCl. However, 80% of the isolates recovered from the control plants do not support the growth at this salt concentration. The R. etli isolates were less tolerant to salt and they did not grow in salt concentrations higher than 0.6% NaCl. 3.4. Effect of water deficiency on nodulation and shoot dry yield under controlled conditions In order to study further the correlation between the in vitro salt tolerance of rhizobia and the symbiotic effectiveness under water deficiency, we used the high-salt tolerant and effective strain E. meliloti 4H41 (4.4% NaCl), previously isolated from common bean grown in the Tunisian oases (Mnasri et al., 2006), and the reference strain CIAT899T (1.8% NaCl). The experiment was


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Table 3 Screening for NaCl tolerance of common bean rhizobia isolated from the two soil samples under control or water-deficient conditions NaCl in the growth medium in % (w/v) Treatment

Species

0.2

0.4

0.6

Isolates from control plants

R. gallicum (19) R. etli (10) S. fredii (8)

3

5

8 2

Isolates from water-deficient plants

0.8

0 3

1

1.2

1.4

1.6

1.8

7

1

1

2

0

2

R. gallicum (8) S. fredii (2)

1 4

1

3 1

2

2

0

0 1

0

Values are numbers of isolates that tolerate NaCl up to the corresponding concentration. The total number of strains tested from each species is given in parentheses.

control 300

b

23%

b c

35%

200

50%

c d

100

d

e

77%

CIAT899 4H41

Both (1/2, 1/2)

CIAT899 4H41

cv. Flamingo

65%

Both (1/2, 1/2)

50%

control

Water deficient

Inoculant a

a

45%

41%

cv. Coco

1

Shoot dry weight (g plant -1)

55%

59% d

c

0

4H41

a

b

Nodule number (plant -1)

CIAT899

Water deficient

a

a

a

a

a

Control

Water deficient

cv. Flamingo

0.8

Control

Water deficient

cv. Coco

Fig. 2. Effect of water deficiency on the nodule occupancy of two common bean cultivars co-inoculated by strains CIAT899T and 4H41.

0.6 b

b

c

c

0.4

3.5. Inoculation yield of strain 4H41 under field conditions

d

d e

0.2

e f

f

0 To

CIAT899 4H41

Both (1/2, 1/2)

cv. Flamingo

To

CIAT899 4H41

Both (1/2, 1/2)

cv. Coco

Fig. 1. Effect of water deficiency on nodulation (a) and shoot dry weight (b) of two common bean cultivars grown on sterilized sand and inoculated with R. tropici CIAT899T and/or E. meliloti bv. mediterranense 4H41. Means assigned with different letters are significantly different for each cultivar (po0.05).

conducted under controlled conditions on sterilized sand. The results are summarized in Fig. 1. The plants inoculated by the strain 4H41 were significantly less affected by water deficiency than the plants inoculated by CIAT899T. The co-inoculated plants under water deficiency were also significantly better than the plants inoculated by CIAT899T alone. Monitoring of the nodule occupancy showed that water deficiency significantly affected the competition between strains CIAT899T and 4H41 (Fig. 2).

The inoculation of four fields with strain 4H41 significantly increased the nodule number, the shoot dry weight and the grain yield of both cultivars used (Table 4). More than twofold increase in grain yield was observed with both cultivars used in the different soils. Nodulation by indigenous rhizobia was observed only in field A which received a complement of irrigation according to plant need. 4. Discussion The objective of the work described in this paper was to study the effect of water deficiency on nodulation, rhizobial diversity and growth of common bean. In the first experiment conducted on soil samples, we showed that water deficiency induced a significant decrease in nodule number and shoot dry yield. Molecular typing of root nodule isolates revealed that the indigenous rhizobia in soil sample A are homogenous and belonged all to R. gallicum, whereas those from soil sample B were composed of R. etli,


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Table 4 Inoculation yields of strain 4H41 under field conditions Site

A C D E

Non-inoculated

Inoculated

Cultivar Nodule number (plant1)

Soot dry weight (g plant1)

Grain yield (g plant1)

Nodule number (plant1)

Soot dry weight (g plant1)

Grain yield (g plant1)

Coco Flamingo Coco Flamingo Coco Flamingo Coco Flamingo

2.9 4.1 2.0 4.0 1.9 2.3 2.0 3.2

5.6 6.6 4.2 7.5 3.5 3.2 Nd Nd

35 40 13 18 9 13 14 16

6.4 10.6 6.2 10.8 3.8 4.5 4.5 5.2

12.0 19.8 11.2 15.2 7.8 6.2 Nd Nd

23 21 0 0 0 0 0 0

Soil A received a supplement of irrigation but soils C, D and E were not irrigated. Values are means of 30 replicates. The inoculated is significantly better than the non-inoculated (po0.05).

R. gallicum, S. fredii and Agrobacterium sp. S. fredii is the specific symbiont of soybean but it has been reported that some strains have the capacity to induce nodules on beans (Herrera-Cervera et al., 1999; Mhamdi et al., 2002). Some agrobacteria were also previously identified among root nodule extracts: however, they failed to re-nodulate their original host when re-examined for nodulation (De Lajudie et al., 1999; Mhamdi et al., 2002; Hameed et al., 2004). Using gus gene labelling, Mhamdi et al. (2005) gave the first evidence that the agrobacteria identified among nodule extracts were indeed recovered from the inside of nodules. When re-isolated from the invaded nodules, these agrobacteria did not show any symbiotic-gene acquisition (Mhamdi et al., 2005). The mechanism by which these isolates integrated into nodules is still not known. Mrabet et al. (2006) showed that these agrobacteria exercise an antagonistic effect against R. gallicum on agar plates and reduce the nodulation by this species in soil. The water deficiency strongly affected the nodulation by R. etli. Analysis of salt tolerance showed that the isolates of these species are the less tolerant to salt. The most tolerant isolates of this species could not grow in media containing more than 0.6% NaCl. We showed that water deficiency favoured the nodulation of common bean by salt tolerant rhizobia. All the isolates recovered under water deficiency could grow in YEM medium supplemented with 1.2% NaCl. The common point between salt stress and drought is the resulting osmotic stress. One of the immediate responses of rhizobia to osmotic stress concerns the morphological changes (Shoushtari and Pepper, 1985; Busse and Bottomley, 1989). The modification of rhizobial cells will eventually lead to a reduction in infection and nodulation of legumes (Hunt et al., 1981; Zahran and Sprent, 1986). Some rhizobia counteract the dehydration effect of low water activity in the medium by accumulating low-molecular-weight organic solutes called osmolytes (Botsford and Lewis, 1990; Smith et al., 1994). Thus, the selection of the most efficient strains among the droughttolerant isolates may contribute to the formulation of

inoculants for the arid regions and, therefore, this commands great interest. Since we know that water deficit also results in restriction of nodule development and function (Serraj et al., 1999; Zahran, 1999), the effectiveness of these inoculants needs to be confirmed. The host plant is also generally more sensitive to osmotic stresses than the microsymbiont (Zahran, 1991). Thus the selection of superior strains risks being ineffective. Could the use of highly osmo-tolerant strains improve the nitrogen fixation at moderate soil moisture within the range of the legume host? In order to investigate this issue, water deficiency was studied on sterilized sand using strains CIAT899T and 4H41. Common bean symbionts are considered very sensitive to salt. The most tolerant strains have been reported to grow in salt concentrations up to 350 mM NaCl, but many strains do not grow in much lower NaCl concentrations (Breedveld et al., 1991; Amarger et al., 1997). R. tropici CIAT899T is able to grow in YEM medium containing up to 300 mM NaCl (1.8%). Similarly, all the isolates recovered in this study under water deficiency could not grow in NaCl concentrations higher than 300 mM. However, other rhizobia from other legumes are more salt tolerant and support the growth in higher saline concentrations. The highest tolerance values were usually found with E. meliloti, up to 750 mM NaCl (Mohammad et al., 1991; Embalomatis et al., 1994; Mnasri et al., 2006). Recently, a highly salt-tolerant strain (strain 4H41) was isolated from common bean nodules from the Tunisian oases (Mnasri et al., 2006). This strain did not nodulate Medicago species but was effective on its original host. The strain 4H41 constituted a novel biovar (bv. mediterranense) inside E. meliloti (formerly Sinorhizobium meliloti). Our results showed that strain 4H41 was more competitive and more effective in nitrogen fixation under water deficiency than strain CIAT899T. These results suggest that the growth of common bean can be improved by inoculating plants with competitive and highly salttolerant rhizobia. This could be an economically feasible


way to increase common bean production in water-limited environments. The field inoculation trials conducted with strain 4H41 induced a significant increase in nodule number, shoot dry weight and grain yield even in the non-irrigated fields. In these soils, the nodulation by indigenous rhizobia was totally absent. However, when common bean was grown on soil samples from these fields and adequately irrigated numerous nodules could be observed (data not shown), suggesting that the native rhizobia were not tolerant to water deficiency. More field inoculation trials are needed to confirm the usefulness of strain 4H41 as inoculant for the semi-arid regions. 5. Conclusions The results of this study showed that water deficiency affected the nodulation, the nitrogen fixation and the genetic diversity of the rhizobia nodulating common bean. Under water deficiency, the host plant is nodulated by the most competitive genotypes among the salt-tolerant strains. This work constitutes the first report of a strain enhancing the nitrogen fixation of common bean under water deficiency. Acknowledgements This work was supported in part by grants from the Aquarhiz project (FP6 INCO-CT-2004). References Amarger, N., Macheret, V., Laguerre, G., 1997. Rhizobium gallicum sp. nov. and Rhizobium giardinii sp. nov. from Phaseolus vulgaris nodules. International Journal of Systematic Bacteriology 47, 996–1006. Botsford, J.L., Lewis, T.A., 1990. Osmoregulation in Rhizobium meliloti: production of glutamic acid in response to osmotic stress. Applied and Environmental Microbiology 56, 488–494. Breedveld, M.W., Zevenhuizen, L.P.T.M., Zehnder, A.J.B., 1991. Osmotically-regulated trehalose accumulation and cyclic beta-(1,2)glucan excreted by Rhizobium leguminosarum bv. trifolii TA-1. Archives of Microbiology 156, 501–506. Busse, M.D., Bottomley, P.J., 1989. Growth and nodulation responses of Rhizobium meliloti to water stress induced by permeating and nonpermeating solutes. Applied and Environmental Microbiology 55, 2431–2436. Buttery, B.R., Park, S.J., Findlay, W.J., 1987. Growth and yield of white bean (Phaseolus vulgaris L.) in response to nitrogen, phosphorus and potassium fertilizer and to inoculation with Rhizobium. Canadian Journal of Plant Science 67, 425–432. De Lajudie, P., Willems, A., Nick, G., Mohamed, S.H., Torck, U., Coopman, R., Filali-Maltouf, A., Kersters, K., Dreyfus, B., Lindstrom, K., Gillis, M., 1999. Agrobacterium bv. 1 strains isolated from nodules of tropical legumes. Systematic and Applied Microbiology 22, 119–132. Embalomatis, A., Papacosta, D.K., Katinakis, P., 1994. Evaluation of Rhizobium meliloti strains isolated from indigenous populations northern Greece. Journal of Agronomy and Crop Science 172, 73–80. Fuhrmann, J., Davey, C.B., Wollum, A.G., 1986. Desiccation tolerance in clover rhizobia in sterile soils. Soil Science Society of America Journal 50, 639–644.

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