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Growth promotion of chickpea and barley by a phosphate solubilizing

strain of Mesorhizobium mediterraneum under growth chamber conditions

A. Peix

a

, A.A. Rivas-Boyero

b

, P.F. Mateos

b

, C. Rodriguez-Barrueco

a

, E. MartõÂnez-Molina

b

,

E. Velazquez

b,

*

a_{Departamento de ProduccioÂn Vegetal. IRNA. CSIC. Salamanca, Spain.}

b_{Departamento de MicrobiologõÂa y GeneÂtica, Facultad de Farmacia, Edi®cio Departamental, Universidad de Salamanca, Salamanca 37007, Spain.}

Received 11 November 1999; received in revised form 27 April 2000; accepted 22 May 2000

Abstract

The ef®cacy of a strain ofMesorhizobium mediterraneumto enhance the growth and phosphorous content in chickpea and barley plants was assessed in a soil with and without the addition of phospates in a growth chamber. The results obtained show that the strain PECA21 was able to mobilize phosphorous ef®ciently in both plants when tricalcium phosphate was added to the soil. In barley and chickpea growing in soils treated with insoluble phosphates and inoculated with strain PECA21 the phosphorous content was signi®cantly increased in a 100 and 125%, respectively. Also, the dry matter, nitrogen, potassium, calcium and magnesium content in both plants was signi®cantly increased in inoculated soil added with insoluble phosphate. These results show that the inoculation of a soil with rhizobia should not be based only on the effectiveness of the strains with respect to their nitrogen ®xation potential, since these microorganisms can increase the growth of plants by means of other mechanisms, for example the phosphate solubilization.q_{2001 Elsevier Science Ltd. All rights reserved.}

Keywords: Phosphate solubilization; Rhizobia; Chickpea; Barley

1. Introduction

The phosphorous is an essential plant nutrient which is added to soil as inorganic phosphates. A large portion of these phosphates used as fertilizers is immobilized after application and becomes unavailable to plants (Dey, 1988; Singh and Kapoor, 1994), although other soil characteristics play also a role in the solubility of applied phosphorous (pH, soil C, etc.). According to the results obtained by Scheffer and Schachtschabel (1992) only 0.1% of the total phosphor-ous from soil is available to plants. Nevertheless, the free inorganic phosphorous in soil solution plays a central role in P-cycling and plant nutrition (Scheffer and Schachtschabel, 1992). Apart from fertilization and enzymatic decomposi-tion of organic compounds, microbial P-mobilizadecomposi-tion would be the only possible way to increase plant-available phos-phorous (Illmer and Schinner, 1992). Because of that, the inoculation of a soil with phosphate solubilizing microor-ganisms may alleviate this problem (Halder et al., 1990a; Ilmer et al., 1995).

For this reason, many authors have isolated solubilizing

microorganisms in different soils (Illmer and Schinner, 1992; Nahas, 1996). Some authors have described the phos-phate solubilization by rhizobia (Halder et al., 1990a,b). According to their results, strains of Rhizobium legumino-sarum biovar viceae and Mesorhizobium sp. nodulating chickpea were the most effective solubilizers in vitro, indi-cating that the solubilization of phosphorous varies in differ-ent strains of the same species.

According to Halder et al. (1990b), the strains isolated from Cicer arietinum were the best solubilizers of phos-phorous in liquid medium. Currently, two species were described as symbionts ofC. arietinum(Nour et al., 1994, 1995) and were reclassi®ed asMesorhizobium(Jarvis et al., 1998). The rapid identi®cation of these species, M. ciceri andM. mediterraneum, is possible based on a new techni-que, Staircase electrophoresis, that allowed the obtention of LMW RNA pro®les (VelaÂzquez et al., 1998a,b).

The LMW RNA pro®les include three zones in prokar-yotes Gram positives as well as Gram negatives: 5S rRNA zone, class 1 tRNA and class 2 tRNA (VelaÂzquez et al., 1998a,b) and four zones in eukaryotes (yeasts): 5.8S rRNA, 5S rRNA, class 1 tRNA and class 2 tRNA (VelaÂz-quez et al., 2000). From these works, at present we can af®rm that all strains belonging to the same species display

PII: S 0 0 3 8 - 0 7 1 7 ( 0 0 ) 0 0 1 2 0 - 6

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* Corresponding author. Fax:134-923-224876.

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the same LMW RNA pro®le (both 5S rRNA zone and tRNA zone) and all species belonging to the same genus display the same 5S rRNA zone. Therefore, different genera can be distinguished by 5S rRNA zone and different species can be distinguished by tRNA zone. In this way, any strain can be identi®ed by comparison of its LMW RNA pro®le with the LMW RNA pro®le of the type strain of a species.

Thus, our objective in this work was the isolation and identi®cation of strains nodulatingCicer which are able to solubilize phosphates, the evaluation of their ability to solu-bilize phosphate in vitro and to mosolu-bilize phosphorous in plant.

2. Materials and methods

2.1. Sample sites and collection of soil samples

Soil samples were taken from a soil in Northern Spain (Pedrosillo el Ralo). This soil, traditionally, has been culti-vated with an autochtonous variety (Pedrosillano) of C. arietinum (chickpea) in alternance with Hordeum vulgare (barley). Soil samples were taken at a depth of 15±20 cm from three sites in the soil. Soil samples were placed in a cool box for transport, stored at 58C, and then used for plant inoculation tests within 2 days of collection. Soil analyses were performed according to the guidelines of the Soil Conservation Service (1972). The soil was classi®ed according to their morphology and analytical data following the US Soil Taxonomy (Soil Survey Staff, 1994). The char-acteristics of the soil are shown in Table 1.

2.2. Counts of phosphate solubilizers

For counts of phosphate solubilizing bacteria we have used the method of Thomas and Shantaram (1986) modi®ed as follows: for each site, the pooled soil was passed through a 2 mm sieve and mixed thoroughly. A 10 g sample from each soil was emulsi®ed in 90 ml of sterile water. Serial decimal dilutions were made from this suspension up to 1:107. Five aliquots of 0.1 ml of each dilution were used to inoculate petri dishes with YED (yeast extract 0.5%; glucose 1% and agar 2%) supplemented with a 0.2% of tricalcium-phosphate (YED-P). The plates were incubated at 288C for 7 days. After this time the number of colonies surrounded by a clear zone indicating the phosphate solubi-lization was counted.

2.3. Evaluation of abundance of rhizobia strains nodulating chickpea

Determination of the most probable number (MPN) of rhizobia was carried out according to method of Brockwell (1982). Soil dilutions were prepared using the methods in Section 2.2. Three 1 ml aliquots were used to inoculate three C. arietinum plants grown axenically in the solution of Rigaud and Puppo (1975). On day 30 after inoculation,

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the nodulated plants in each of the dilutions were counted and the MPN was calculated following the method of Brockwell (1982).

2.4. Isolation of strains nodulating chickpeas

Surface-sterilized seeds ofC. arietinumvar. Pedrosillano were germinated axenically on water agar and seedlings were transferred to pots with soil from Pedrosillo el Ralo for 30 days. Each pot (14 cm diameter) contained 2 kg of soil and ®ve plants. The pots were placed in a plant growth chamber with mixed incandescent and ¯uorescent lighting (400 microeinsteins m22s21; 400±700 nm), programmed for a 16 h photoperiod, day±night cycle, with a constant temperature varying from 15±27₈C (night±day), and 50± 60% relative humidity. Several strains nodulating chickpea were isolated from different plants on YMA dishes (Vincent, 1970) and six rhizobial strains phosphate solubi-lizing were selected for this study.

2.5. Evaluation of tricalcium phosphate solubilization of rhizobial strains

The ability to solubilize tricalcium-phosphate of the type strains of several species of rhizobia belonging to different genera and of the rhizobial isolates in the present study (see Table 2) was tested in petri dishes containing YED (yeast extract 0.5%; glucose 1% and agar 2%) supplemented with a 0.2% of tricalcium-phosphate (YED-P). A suspension of each strain was inoculated in this medium and the plates

were incubated for 7 days until the solubilization zone surrounding the colonies was observed. The criterium for strain selection was the diameter of clearing zone surround-ing the colonies of each strain (de Freitas et al., 1997).

2.6. RNA extraction and LMW RNA pro®le analysis

For the LMW RNA pro®ling, the strains isolated in this study and the type strains of the two species nodulating chickpea, M. ciceri USDA 3383T and M. mediterraneum USDA 3392T, were used.

The LMW RNA of the strains studied was extracted using the method of HoÈ¯e (1988) and prepared as reported else-where (Cruz-SaÂnchez et al., 1997). LMW RNA pro®les were examined using Staircase Electrophoresis in 14% polyacrylamide gels under denaturing conditions in steps of 10 min, rising through a constant ramp with 50 V increases from 100 to 2300 V (Cruz-SaÂnchez et al., 1997). The following commercial molecules from Boehringer Manheim (Manheim, Germany) and Sigma (St. Louis, MO, USA) were used as reference: 5S rRNA from Esche-richia coliMRE 600 (120 and 115 nucleotides) (Bidle and Fletcher, 1995), tRNA speci®c for tyrosine fromE. coli(85 nucleotides) and tRNA speci®c for valine fromE. coli(77 nucleotides) (Sprinzl et al., 1985). After electrophoresis, the gels were silver-stained as described by Haas et al. (1994).

2.7. Mobilization of phosphorous in plants

Experiments for studying the phosphorous mobilization A. Peix et al. / Soil Biology & Biochemistry 33 (2001) 103±110

Table 2

Phosphate solubilization in plate containing YED-P by type strains of species of the family rhizobiaceae and by the strains isolated in this study (T: type strain)

Strain Phosphate solubilization Host plant Geographic origin Source

Bradyrhizobium japonicum

ATCC 10324T 2

Glycine max Japan Jordan (1982)

Rhizobium leguminosarumbio

varviceaeATCC 10004T 2

Pisum sativum USA Skerman et al. (1980)

Rhizobium tropiciIIB

CIAT 899T 2

Phaseolus vulgaris Colombia MartõÂ nez-Romero et al. (1991)

Sinorhizobium fredii ATCC 35423T

2 Glycine max China Chen et al. (1988)

Sinorhizobium meliloti USDA 1002T

2 Medicago sativa USA de Lajudie et al. (1994)

Mesorhizobium ciceri USDA 3383T

1 Cicer arietinum Spain Nour et al. (1994)

Mesorhizobium mediterraneum USDA 3392T

1 Cicer arietinum Spain Nour et al., (1995)

Mesorhizobium mediterraneum PECA09

1 Cicer arietinum Spain This study

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in plants were made on chickpea and barley and were conducted in pots containing soil from Pedrosillo el Ralo (Spain). Each pot (14 cm diameter) contained 2 kg of soil. The pots were placed in a plant growth chamber in the conditions described in Section 2.4. The experimental design was performed as follows Ð treatment 1: uninocu-lated soil (control soil); treatment 2: uninocuuninocu-lated soil with addition of insoluble phosphate (Ca3PO40.2%); treatment 3: uninoculated soil with addition of soluble phosphate (K2HPO40.1% and KH2PO40.1%); treatment 4: soil inocu-lated with strain PECA21 and treatment 5: soil inocuinocu-lated with strain PECA21 with addition of insoluble phosphate (Ca3PO40.2%). The insoluble and soluble phosphates were mixed thoroughly with the soil in a plastic bag before use. Five pots were used for each treatment. Five seeds were placed in each pot at 2 cm depth.

For inoculation, strain PECA21 was grown in plate dishes with YMA for 5 days. After that, sterile water was added to the plates in order to obtain a suspension with approxi-mately 1011cells/ml. For inoculation, we have added 1 ml from the suspension of strain PECA 21 to each seed placed in the pot.

At harvest (40 days) the length and the dry weight of the aerial part of the inoculated plants of chickpea and barley were determined. Plant nitrogen, phosphorous, potassium, calcium and magnesium content was measured according to methods of the Association of Of®cial Analytical Chemists (1990). The data obtained were analyzed by one-way analy-sis of variance, with the mean values compared using the Fisher's Protected LSD (Low Signi®cative Differences)

p0:05:

3. Results

3.1. Counts of phosphate solubilizers and rhizobia nodulating chickpea

The number of bacteria phosphate solubilizers in the soil studied was lower than 1£102cells/g of soil. Also, the MPN obtained for rhizobia nodulating chickpea was lower than 1£102cells/g (see Table 1). In both cases the number of solubilizing or nodulating bacteria is out of the detection limit of count techniques.

3.2. Evaluation of tricalcium phosphate solubilization of rhizobial strains

The results for the detection of phosphate solubilization in the species from the Family Rhizobiaceae able to induce nodules in legumes are shown in Table 2. According to the results obtained, only the strains nodulating chickpea (including the type strains) are able to solubilize phosphate in the medium used in this study. In our study, the strain PECA21 was the fastest solubilizer in petri dishes. This strain produced the largest zone of clearing ($15 mm) in

7 days in YED-P plates (de Freitas et al., 1997) and hence was selected for the inoculation tests.

3.3. RNA extraction and LMW RNA pro®le analysis

The identi®cation of the strains isolated in this study was made by using the LMW RNA pro®ling. Fig. 1 shows the LMW RNA pro®le of the type strains nodulating chickpea, M. ciceri (Lane 1), M. mediterraneum (Lane 2) and the strains isolated in this study (lanes 3±8). As may be observed in this ®gure, the strains PECA 09, PECA20, PECA21, PECA22 and PECA30 (lanes 4±8) display iden-tical LMW RNA pro®le as the type strain ofM. mediterra-neum and the strain PECA26 (lane 3) displays the same LMW RNA pro®le asM. ciceri.

3.4. Mobilization of phosphorous in plants

The results of the inoculation assays are shown in Table 3. According to these results, a signi®cant increase of most parameters measured in this study was observed when the soil was inoculated with strain PECA21 compared to the soil without inoculum, either in barley and in chickpea plants. The highest results were obtained in the plants grown in soil with strain PECA21 and insoluble phosphate, except P content value, which was higher in both plants when soluble phosphorous was added to the soil.

In barley plants, dry weight was signi®cantly increased in a 43%p_,0:05when the soil was inoculated with strain PECA21 compared to the soil without inoculum. Insoluble phosphate addition to the inoculated soil increased signi®-cantly the dry matter in a 9% with respect to the previous case. Hence, the dry matter total increase in the soil with insoluble phosphate and inoculated with strain PECA21 was more than 50% with respect to the uninoculated soil.

Although the P-uptake by barley plants was higher in soils with soluble phosphate than in inoculated soils, the phosphorous content in barley plants grown in soil inocu-lated with PECA21 was signi®cantlyp_,0:05increased in a 92% compared to the plants growing in uninoculated soil and for the plants grown in soils treated with insoluble phosphates and inoculated with strain PECA21 the phos-phorous content was increased in a 100%p_,0:05:

The nitrogen content per plant in barley was also signi®-cantly increased in soils inoculated with PECA21 p_, 0:05: The nitrogen content in soils inoculated with this strain and added with insoluble phosphate was increased in a 100% compared to the uninoculated soil. Also, Ca, K and Mg content values were higher in inoculated soil than in uninoculated soil.

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insoluble phosphate and inoculated with strain PECA21 was 18% higher with respect to the uninoculated soil.

Although the phosphorous content in chickpea plants was higher in soils with soluble phosphate than in inoculated soils like in barley plants case, the phosphor-ous content in chickpea plants growing in soil inocu-lated with PECA21 was signi®cantly p_,0:05 increased in a 27% compared to the plants grown in uninoculated soil, and for the plants growing in soils treated with insoluble phosphates and inoculated with strain PECA21 the phosphorous content was increased in a 125% p,0:05:

The Nitrogen content per plant in chickpea plants was also signi®cantly increased in soils inoculated with PECA21 p,0:05: The nitrogen content in soils inoculated with this strain and added with insoluble phosphate is increased in a 83% compared to the unin-oculated soil. As in barley case, in chickpea, Ca, K and Mg values were higher in inoculated soil than in unin-oculated soil.

Thus, the results obtained show that the chickpea and barley plants grown in soil additioned with insoluble phos-phate and inoculated with Mesorhizobium mediterraneum PECA21 strain have signi®cantly p,0:0:5 higher dry matter, phosphorous, nitrogen, potassium, calcium and magnesium content with respect to uninoculated soils.

4. Discussion

The phosphate solubilizing microorganisms have been considered as PGPR (Plant Growth Promoting Rhizobac-teria), although their role on plant growth is a subject of controversy. Some authors think that the inoculation of soil with phosphate solubilizing microorganisms can increase the crops yield by other mechanisms, such as the growth factor production (Tinker, 1980). Other studies have shown that the inoculation of soil with phosphate solubiliz-ing microorganisms yields crops similar to those obtained by addition of soluble phosphorous (Ralston and McBride, 1976).

Several species and genera of bacteria have been reported as being able to solubilize phosphates. Halder et al. (1990b) have found that the strains isolated fromCicerwere the best phosphate solubilizers within rhizobia. In our study, the rhizobia strains that were the best phosphate solubilizers in YED1tricalcium phosphate plates could also nodulate chickpeas. The type strains ofM. ciceriandM. mediterra-neumas well as the strains isolated fromCicerin this study were able to solubilize phosphate in vitro. Until 1995, all the strains isolated fromCicerwere considered to be included in the species Rhizobium loti. However, currently, two species nodulating chickpea, M. ciceri and M. mediterra-neum, are accepted (Nour et al., 1994, 1995; Jarvis et al., 1998). Therefore, it is now necessary to identify the strains isolated fromCicerwith one of the two species cited above. We have identi®ed the strains isolated from chickpea in this study using a new electrophoresis technique, Staircase electrophoresis, that allows the optimal separation of stable Low Molecular Weight (LMW) RNA, which include the tRNAs and the 5S rRNA. VelaÂzquez et al. (1998a,b) have shown in a previous analysis of LMW RNA for members of the family Rhizobiaceae that each genus of this Family shows a characteristic and unique 5S rRNA pro®le (all species belonging to the same genus display the same 5S rRNA pro®le), and each species shows a characteristic and unique tRNA pro®le (all strains belonging to the same species display the same tRNA pro®le). In the same study, we have demonstrated that the two species nodulating chickpea,M. mediterranuemandM. cicerishows a different tRNA pro®le, thus each species have a characteristic and unique tRNA pro®le which allows to differentiate them. In other studies we have demonstrated that the strains of the same microbial species have the same LMW RNA pro®le (VelaÂzquez et al., 1998a,b; VelaÂzquez et al., 2000). There-fore, LMW RNA pro®les allow the rapid identi®cation of any rhizobial strain and we have used them for rapid iden-ti®cation of isolates from chickpea in the present study.

According to the results obtained, ®ve of the strains (PECA09, PECA20, PECA21, PECA22 and PECA30) isolated in this study have the characteristic LMW RNA pro®le ofM. mediterraneumand the strain PECA26 showed the characteristic pro®le ofM. ciceri. These results are in agreement with the results obtained by other authors (Nour A. Peix et al. / Soil Biology & Biochemistry 33 (2001) 103±110

Fig. 1. LMW RNA pro®les of the strains of the species from the Family Rhizobiaceae included in this study. Lane (1)M. ciceriUSDA 3383T_{, (2)}

M. mediterraneumUSDA 3392T_{, (3)}_{M. ciceri}_{PECA 26, (4)}_M.

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et al., 1994, 1995) who have shown that the two species are represented among nodulating chickpea strains isolated in Spain.

The strain M. mediterraneumPECA21, as it has been pointed out in Section 3.2, was the best phosphate solubiliz-ing in plates. Thus, this strain was selected for studies of phosphorous mobilization in plants. Traditionally, the bene-®cial effect of rhizobia as nitrogen ®xers in symbiosis with legumes has been reported, however, the effect of these microorganisms in the phosphorous mobilization in plants has been less studied, and this effect can be shown in legumes as well as in other plants. The works of Antoun et al. (1998) and Chabot et al. (1996, 1998) have demon-strated that phosphate solubilizing strains ofRhizobiumand Bradyrhizobium increase growth in maize, lettuce and radishes. According to Chabot et al. (1996) the P solubiliza-tion effect seems to be the most important mechanism of plant growth promotion and they have demonstrated that two phosphate solubilizing strains of R. leguminosarum bv.phaseoliincreased the P content in maize and lettuce.

The strains of rhizobia included in this study have been isolated from a soil of Pedrosillo el Ralo (Spain). This soil, traditionally, has been cultivated with an autochtonous vari-ety (Pedrosillano) ofC. arietinum(chickpea) in crop rota-tion withH. vulgare(barley). This soil is placed in a Spanish region which has extensive crops of chickpea. All soils in this region have little amounts of phosphate, because of that, in our experiment we have included a soil with addition of soluble phosphorous to increase the P-available in this soil. Moreover, the soil from Pedrosillo have a low content in rhizobia nodulating chickpea and also in phosphate solubi-lizing bacteria. These two characteristics allow us to detect the effect of the inoculation of soil with a phosphate solu-bilizing rhizobial strain. In this study we have used the two plants that are currently cultivated in the poor soils in Spain, a legume (C. arietinum, chickpea) and a cereal (H. vulgare, barley).

According to the results obtained, in uninoculated soils, the total phosphorous per plant is greatly increased if solu-ble phosphate is added, but this increment is higher when the soil is also inoculated with the strain PECA21. Although, in soils inoculated with PECA21 strain plus Ca3PO4 the total phosphorous per plant is lower than in soil added with soluble phosphorous, the plants have a higher nitrogen content and a signi®cantly higher dry weight.

The same effect was found by other authors in nonle-gumes (Antoun et al., 1998; Chabot et al., 1996, 1998), but in these studies there are no data on nitrogen content of inoculated plants. In our work we have founded that the nitrogen content is increased in chickpea plants grown in inoculated soils with PECA21, but also in barley plants and this effect is very important because the increase may be 100%. The lack of fertilization of the soil allowed us to measure this effect which may be due to two reasons: (i) an increase in soil N-uptake because of a better development

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of the plant when having higher amount of available P (through the same mechanism Ca, K and Mg increase could be explained) and (ii) by nitrogen ®xation in barley roots by strain PECA21. This second mechanism has been already reported for roots of rice cultivated in river Nilo Delta whose crop is carried out with clover in crop rotation. There are references about an increase in Nitrogen content of rice plants inoculated with strains ofR. leguminosarum bv trifolii when this microorganism gets into the roots (Yanni et al. 1997). However, to con®rm this hypothesis in barley, in the future would be necessary to perform microscopic studies in the same way as they were performed for the rice case.

Rhizobia have been extensively studied as nitrogen ®xers with legumes and only few studies have demonstrated that are able to enhance the growth of nonlegumes. In this work, we have demonstrated that a solubilizing phosphates strain ofM. mediterraneumcan enhance the growth of chickpea (a legume) and barley (a cereal) and that the inoculation of seeds with rhizobia can be bene®cial for plants used in crop rotation with legumes. Rhizobia strains show advan-tages with respect to other microorganisms to be used as inoculants, because a great number of studies on infection process molecular aspects have been performed and there is a great experience in seed inoculation used in extensive crops (soya, for example). A careful selection of rhizobial strains able to ®x nitrogen and mobilize phosphorous may allow an adequate inoculation of seeds and a signi®cant increase of legume and nonlegume crops.

Acknowledgements

This work was supported by the Junta de Castilla y LeoÂn and the DGICYT (DireccioÂn General de InvestigacioÂn Cien-tõÂ®ca y TeÂcnica). The authors thank Imelda Geldart for revising the English version of the manuscript. We are also grateful to the soil analysis service staff from IRNA for their help in this work.

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