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Soil Biology & Biochemistry 33 (2001) 103±110
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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,*
b

a
Departamento de ProduccioÂn Vegetal. IRNA. CSIC. Salamanca, Spain.
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 of Mesorhizobium mediterraneum to 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 phosphorous 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 decomposition of organic compounds, microbial P-mobilization would
be the only possible way to increase plant-available phosphorous (Illmer and Schinner, 1992). Because of that, the
inoculation of a soil with phosphate solubilizing microorganisms may alleviate this problem (Halder et al., 1990a;
Ilmer et al., 1995).

For this reason, many authors have isolated solubilizing
* Corresponding author. Fax: 134-923-224876.
E-mail address: evp@gugu.usal.es (E. Velazquez).

microorganisms in different soils (Illmer and Schinner,
1992; Nahas, 1996). Some authors have described the phosphate solubilization by rhizobia (Halder et al., 1990a,b).
According to their results, strains of Rhizobium leguminosarum biovar viceae and Mesorhizobium sp. nodulating
chickpea were the most effective solubilizers in vitro, indicating that the solubilization of phosphorous varies in different strains of the same species.
According to Halder et al. (1990b), the strains isolated
from Cicer arietinum were the best solubilizers of phosphorous in liquid medium. Currently, two species were
described as symbionts of C. arietinum (Nour et al., 1994,
1995) and were reclassi®ed as Mesorhizobium (Jarvis et al.,
1998). The rapid identi®cation of these species, M. ciceri
and M. mediterraneum, is possible based on a new technique, 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 prokaryotes 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 (VelaÂzquez et al., 2000). From these works, at present we can
af®rm that all strains belonging to the same species display

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104

2.19

, 10 2

, 10 2

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 nodulating Cicer which are able to
solubilize phosphates, the evaluation of their ability to solubilize phosphate in vitro and to mobilize phosphorous in
plant.
2. Materials and methods
2.1. Sample sites and collection of soil samples

0.08

2.6

14.90

1.14

Soil samples were taken from a soil in Northern Spain
(Pedrosillo el Ralo). This soil, traditionally, has been cultivated 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 58 C, 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 characteristics of the soil are shown in Table 1.
2.2. Counts of phosphate solubilizers

a

b

rhizobianodulating chickpea MPN.
Phosphate Solubilizing Bacteria count.

1.86
Pedrosillo Calcic
Loamy 1 year with chickpea 7.9
el Ralo Rhodoxeralf Clayey 1 year with barley

PH water Organic matter (%) Total N (%) Available P (mg kg 21) Total K (%) CaCO3 (mg kg 21) Mg (cmol kg 21) RMPN (cells/g) a PSBC (cells/g) b

Texture Planting history
Type
Site

Table 1
Characteristics of the soil from Pedrosillo el Ralo (Spain) used in this study

A. Peix et al. / Soil Biology & Biochemistry 33 (2001) 103±110

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
1:10 7. 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 288 C for 7 days. After this time the number of colonies

surrounded by a clear zone indicating the phosphate solubilization 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,

105

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 10324 T
Rhizobium leguminosarum bio
var viceae ATCC 10004 T
Rhizobium tropici IIB
CIAT 899 T
Sinorhizobium fredii
ATCC 35423 T
Sinorhizobium meliloti
USDA 1002 T
Mesorhizobium ciceri
USDA 3383 T
Mesorhizobium mediterraneum
USDA 3392 T

Mesorhizobium mediterraneum
PECA09
Mesorhizobium mediterraneum
PECA20
Mesorhizobium mediterraneum
PECA21
Mesorhizobium mediterraneum
PECA22
Mesorhizobium mediterraneum
PECA30
Mesorhizobium ciceri PECA26

2

Glycine max

Japan

Jordan (1982)

2

Pisum sativum

USA

Skerman et al. (1980)

2

Phaseolus vulgaris

Colombia

MartõÂ nez-Romero et al. (1991)

2

Glycine max

China

Chen et al. (1988)

2

Medicago sativa

USA

de Lajudie et al. (1994)

1

Cicer arietinum

Spain

Nour et al. (1994)

1

Cicer arietinum

Spain

Nour et al., (1995)

1

Cicer arietinum

Spain

This study

1

Cicer arietinum

Spain

This study

1

Cicer arietinum

Spain

This study

1

Cicer arietinum

Spain

This study

1

Cicer arietinum

Spain

This study

1

Cicer arietinum

Spain

This study

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

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 surrounding the colonies of each strain (de Freitas et al., 1997).

2.4. Isolation of strains nodulating chickpeas
Surface-sterilized seeds of C. arietinum var. 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 m 22 s 21; 400±700 nm), programmed
for a 16 h photoperiod, day±night cycle, with a constant
temperature varying from 15±278 C (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 solubilizing 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

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 3383 T and M. mediterraneum
USDA 3392 T, were used.
The LMW RNA of the strains studied was extracted using
the method of Hoȯe (1988) and prepared as reported elsewhere (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 Escherichia coli MRE 600 (120 and 115 nucleotides) (Bidle and
Fletcher, 1995), tRNA speci®c for tyrosine from E. coli (85
nucleotides) and tRNA speci®c for valine from E. 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

106

A. Peix et al. / Soil Biology & Biochemistry 33 (2001) 103±110

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: uninoculated soil (control soil); treatment 2: uninoculated soil with
addition of insoluble phosphate (Ca3PO4 0.2%); treatment 3:
uninoculated soil with addition of soluble phosphate
(K2HPO4 0.1% and KH2PO4 0.1%); treatment 4: soil inoculated with strain PECA21 and treatment 5: soil inoculated
with strain PECA21 with addition of insoluble phosphate
(Ca3PO4 0.2%). 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 approximately 10 11 cells/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 analysis of variance, with the mean values compared using the
Fisher's Protected LSD (Low Signi®cative Differences)
…p ˆ 0:05†:

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 £ 10 2 cells/g of soil. Also, the
MPN obtained for rhizobia nodulating chickpea was lower
than 1 £ 10 2 cells/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 identical LMW RNA pro®le as the type strain of M. mediterraneum and the strain PECA26 (lane 3) displays the same
LMW RNA pro®le as M. 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 , 0:05† when 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 inoculated with PECA21 was signi®cantly …p , 0:05† increased
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 phosphorous 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.
In chickpea plants, dry weight was signi®cantly increased
in a 14% …p , 0:05† when the soil was inoculated with
strain PECA21 compared to the uninoculated soil. Insoluble
phosphate addition to the inoculated soil increased signi®cantly the dry matter in a 4% with respect to the previous
case. Hence, dry matter total increase in the soil with

A. Peix et al. / Soil Biology & Biochemistry 33 (2001) 103±110

107

4. Discussion

Fig. 1. LMW RNA pro®les of the strains of the species from the Family
Rhizobiaceae included in this study. Lane (1) M. ciceri USDA 3383 T, (2)
M. mediterraneum USDA 3392 T, (3) M. ciceriPECA 26, (4) M. mediterraneumPECA 09, (5) M. mediterraneumPECA20, (6) M. mediterraneumPECA21, (7) M. mediterraneumPECA22 and (8) M. mediterraneumPECA30.

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 phosphorous content in chickpea plants growing in soil inoculated 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 uninoculated soil. As in barley case, in chickpea, Ca, K and
Mg values were higher in inoculated soil than in uninoculated soil.
Thus, the results obtained show that the chickpea and
barley plants grown in soil additioned with insoluble phosphate 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.

The phosphate solubilizing microorganisms have been
considered as PGPR (Plant Growth Promoting Rhizobacteria), 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 solubilizing 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 from Cicer were the best
phosphate solubilizers within rhizobia. In our study, the
rhizobia strains that were the best phosphate solubilizers
in YED 1 tricalcium phosphate plates could also nodulate
chickpeas. The type strains of M. ciceri and M. mediterraneum as well as the strains isolated from Cicer in this study
were able to solubilize phosphate in vitro. Until 1995, all the
strains isolated from Cicer were considered to be included
in the species Rhizobium loti. However, currently, two
species nodulating chickpea, M. ciceri and M. mediterraneum, are accepted (Nour et al., 1994, 1995; Jarvis et al.,
1998). Therefore, it is now necessary to identify the strains
isolated from Cicer with 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. mediterranuem and M. ciceri shows 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). Therefore, LMW RNA pro®les allow the rapid identi®cation of
any rhizobial strain and we have used them for rapid identi®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 of M. mediterraneum and the strain PECA26 showed
the characteristic pro®le of M. ciceri. These results are in
agreement with the results obtained by other authors (Nour

108

0.7 ab
0.6 ab
0.9 cde
0.9 cde
0.9 cde
3a
3.5 b
4.7 cd
4.3 cd
5.2 e
2.8 a
7.2 b
8.8 cd
9.3 cd
10.2 e
74.9 a
128 c
190.0 e
95.1 b
168.7 d
4.2 a
6.8 b
7.0 bc
7.4 bcd
7.7 d
23.3 bc
25.9 d
22.1 abc
22.6 abc
25.9 d
Chickpea
Uninoculated soil
Uninoculated soil 1 Ca3PO4
Uninoculated soil 1 soluble P
Inoculated soil with PECA21
Inoculated soil with PECA21
1 Ca3PO4

203.1 ab
205.6 abc
217.2 abc
231.5 cd
240 d

0.07 abc
0.06 ab
0.08 bc
0.11 d
0.14 e
0.15 ab
0.18 bc
0.17 ab
0.37 de
0.37 de
1.8 ab
1.8 ab
2.7 c
3.1 d
3.5 e
5.3 b
4.0 a
14.1 e
10.2 cd
10.6 cd
0.6 ab
0.7 ab
0.7 ab
1.0 c
1.2 d
37.6 ab
33.9 ab
36.1 ab
53.7 c
58.8 d
23.2 ab
24.7 abc
26.0 bc
27.9 d
30.3 e
Barley
Uninoculated soil
Uninoculated soil 1 Ca3PO4
Uninoculated soil 1 soluble P
Inoculated soil with PECA21
Inoculated soil with PECA21
1 Ca3PO4

Total Mg/plant (m g)
Total Ca/plant (m g)
Total K/plant (m g)
Total P/plant (m g)
Total N/plant (mg)
Dry weight (mg)
Aerial height (cm)

Table 3
Effect of inoculation of soil with M. mediterraneum PECA21 on chickpea and barley plants (values followed by the same letter are not signi®cantly different from each other at p ˆ 0:05 according to Fisher's
Protected LSD)

A. Peix et al. / Soil Biology & Biochemistry 33 (2001) 103±110

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 solubilizing 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 demonstrated that phosphate solubilizing strains of Rhizobiumand
Bradyrhizobium increase growth in maize, lettuce and
radishes. According to Chabot et al. (1996) the P solubilization 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. phaseoli increased 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 variety (Pedrosillano) of C. arietinum (chickpea) in crop rotation with H. 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 solubilizing bacteria. These two characteristics allow us to detect
the effect of the inoculation of soil with a phosphate solubilizing 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 soluble 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 nonlegumes (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

A. Peix et al. / Soil Biology & Biochemistry 33 (2001) 103±110

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 of R. 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
of M. mediterraneum can 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 advantages 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 Cientõ®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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