Advances in Environmental Research 6 (2002) 391–399

Alfalfa growth promotion by bacteria grown under iron limiting

conditions
˜ a , J. Juarez
G. Carrillo-Castaneda ´ ˜ a, J.R. Peralta-Videab, E. Gomezb, K.J. Tiemannb,
Munos
M. Duarte-Gardeac, J.L. Gardea-Torresdeyb,*
a
Colegio de Postgraduados, Montecillo, Mexico 56230
b
Department of Chemistry and Environmental Science and Engineering, The University of Texas at El Paso, El Paso,
TX 79968-0513, USA
c
School of Allied Health, The University of Texas at El Paso, El Paso, TX 79968-0513, USA

Accepted 5 March 2002

Abstract

This investigation has been conducted to compare and contrast the impact of 18 potentially plant growth-promoting
bacterial strains, cultured in rich and iron-deficient minimal media, on alfalfa seed germination and seedling vigor.
Our results showed that seed germination was improved by all of the bacterial strains grown in iron-deficient minimal
medium. We also found that all the seeds inoculated with the bacterial strains grown in iron-deficient minimal
medium produced seedlings with larger roots (55–66 mm) than the roots of uninoculated control plants (54.9 mm).
Furthermore, most of the strains cultivated in iron-deficient minimal medium generated seedlings with larger roots
than those seeds inoculated with microbial cells grown in iron-rich medium (37–59 mm). Overall, seeds inoculated
with the strain U cultivated in iron-deficient minimal medium gave rise to plantlets with the greatest length (81 mm).
On the other hand, seeds inoculated with bacterial cells grown under iron-rich media show significantly higher stem
and root dry weight, which is an indication of the influence of the growth conditions on the plant growth-promoting
ability of these microorganisms. 䊚 2002 Elsevier Science Ltd. All rights reserved.

Keywords: Bacterial-enhanced plant growth; Alfalfa

1. Introduction and metal toxicity, as well as by diseases. Previous

Alfalfa is the most important forage crop in the
world. The seed germination and vigor condition of
alfalfa seedlings are characteristics that determine plant
establishment and initial crop development. However,
optimal seed performance and seedling development
often shows high variation in many different alfalfa
varieties. Frequently, plant development is hampered by
environmental conditions, such as metal deficiencies
*Corresponding author. Tel.: q1-915-747-5359; fax: q1-
915-747-5748.
E-mail address: jgardea@utep.edu (J.L. Gardea-Torresdey).
investigations have shown that common root-colonizing
bacteria are plant growth-promoting microorganisms that
can improve plant development under a variety of
environmental conditions (Reynders and Vlassik, 1982;
Kapulnik et al., 1985; Sarig et al., 1988); however, the
known mechanisms by which they promote plant growth
are quite variable. Some of them promote plant growth
mediated by their production of plant regulators (Mor-
dukhava et al., 1991). Other microorganisms, such as
Rhizobium and Azospirillum spp. are able to fix atmos-
pheric nitrogen. Species of Pseudomonas biosynthesize
a variety of chemical compounds with demonstrated
agroactive importance in the biological control of plant

1093-0191/02/$ - see front matter 䊚 2002 Elsevier Science Ltd. All rights reserved.
PII: S 1 0 9 3 - 0 1 9 1 Ž 0 2 . 0 0 0 5 4 - 0
392 ˜
G. Carrillo-Castaneda et al. / Advances in Environmental Research 6 (2002) 391–399

root diseases (Dune et al., 1997). Some of these Table 1

chemical compounds produced by microorganisms in Relation and source of bacterial strains utilized in the present
the rhizosphere may in turn increase the availability and study
uptake of certain essential minerals, such as iron (Imsan-
No Strain Source
de, 1998; Hemming, 1986; Jurkevitch et al., 1988;
Loper and Buyer, 1991; Fabiano et al., 1994; Carrillo- Pseudomonas
˜
Castaneda and Alvarado, 2000). Plants such as oats are 1 P. putida (Pp) IREGEP
able to assimilate iron via siderophores (metal-chelating 2 P. fluorescens (Pf) IREGEP
compounds) (Crowley et al., 1988; Bar-Ness et al., 3 A7 IREGEP
1992; Kawai et al., 1998; Marschner et al., 1986). 4 A9 IREGEP
5 A9m IREGEP
Ferrichrome, a prototype of the hydroxamate siderop-
6 Avm IREGEP
hores, chelates aluminum, gallium, chromium, copper 7 Tb Amman, Jordan
and iron. It has been demonstrated that plants are able 8 Ts Amman, Jordan
to use the hydroxamate-type siderophores ferrichrome, 9 U Amman, Jordan
rodotorulic acid and ferrioxamine B; the catechol-type Rhizobium leguminosarum bv
siderophores, agrobactin; and the mixed ligand cate- phaseoli
chol–hydroxamate–hydroxy acid siderophores biosyn- 10 CP Mex. 19 CEDAF
thesized by saprophytic root-colonizing bacteria 11 CP Mex. 44 CEDAF
(Jurkevitch et al., 1986; Neilands and Leong, 1986; 12 CP Mex. 46 CEDAF
Imsande, 1998). All of these compounds are produced Rhizobium spp.
13 Lc Montevideo, Uruguay
by rhizospheric bacterial strains, which have simple
14 Ls Montevideo, Uruguay
nutritional requirements, and are found in nature in Azospirillum lipopherum
soils, foliage, fresh water, sediments and seawater 15 Sp7 IREGEP
(O’Sullivan and O’Gara, 1992). A particular bacterial 16 Sp59 IREGEP
strain may express any one or more of these traits, 17 UAP40 AUP
depending on environmental conditions, and the impact 18 UAP154 AUP
of the bacterial cells on the plant may vary with the 19 H2O (control)
plant genotype, the presence of the native microflora, ´
IREGEP, Instituto de Recursos Geneticos y Productividad,
and the soil chemical and physical properties. ´
Colegio de Postgraduados, Montecillo, Mexico, CP 56230;
Several investigators have evaluated the possibility of CEDAF, Centro de Edafologıa, ´ Colegio de Postgraduados,
iron acquisition by higher plants from microbial chelated ´
Mexico, CP 56230; and AUP, Autonomous University of Pueb-
iron (Romheld
¨ and Marschner, 1986a,b; Bar-Ness et al., ´
la, Puebla, Mexico.
1991; Yehuda et al., 1996). Surprisingly, no attempts
have been made to determine at an early stage of (Carrillo-Castaneda
˜ and Alvarado, 2000). Our hypoth-
development the extent of plant growth enhancement esis is that the bacterial iron culture conditions at the
and developmental modification of the plants. In previ- time of seed inoculation will determine their promoting
ous studies, it has been demonstrated that strains of potential on early alfalfa seedling development, partic-
Pseudomonas, Rhizobium and Azospirillum respond to ularly root development. The aim of this work was to
iron limitation by secreting siderophores. We have found demonstrate the potential plant growth-promoting effects
that the strains developed under iron limiting conditions of 18 different bacterial strains on alfalfa seed germi-
expelled fluorescent siderophore pigments or non-fluo- nation and seedling vigor at a very early stage of
rescent pigments in the culture medium. This expression development. We believe that growth promotion
is repressed when the microorganisms are developed in depends on the media’s iron concentration (presence or
iron-rich culture media. In addition, in two sets of absence of iron) where the bacterial cultures are devel-
different biological assays, the effectiveness of these oped. This information will be useful for the develop-
exudates in improving plant growth and hindering path- ment of procedures to selectively enhance alfalfa
ogenic bacterial growth has been demonstrated (Carril- seedling root development.
˜
lo-Castaneda ˜
and Peralta, 1988; Carrillo-Castaneda and
´
Vazquez, ˜
1992; Peralta and Carrillo-Castaneda, 1988a,b; 2. Materials and methods
´
Martınez ˜
and Carrillo-Castaneda, 1990). Furthermore,
the ability of these microorganisms to produce siderop- 2.1. Media and bacterial strains
hores was demonstrated by means of a universal color-
imetric assay for the detection and determination of The bacterial strains utilized in this study are listed
siderophores (Schwyn and Neilands, 1987). Recently, in Table 1. These bacterial strains were selected because
we have characterized siderophore-mediated iron trans- in previous studies they were found to produce sider-
port in strains of Rhizobium leguminosarum bv phaseoli ophore-like compounds when grown under iron-defi-
 ˜
G. Carrillo-Castaneda et al. / Advances in Environmental Research 6 (2002) 391–399
cient conditions. These microorganisms were grown in
iron-rich media King’s B and YMB (Vincent, 1975), as
well as in iron-deficient minimal medium (RM-Fe)
(Carrillo-Castaneda
˜ and Peralta, 1988). Solid media
were prepared by adding agar at 1.8% (wyv).
2.2. Bacterial suspension preparation
The microorganisms used to inoculate the seeds were
cultured in liquid media. Series of overnight cultures (3
ml) were prepared as follows: Pseudomonas strains, as
well as Sp 7 and UAP 40, were grown in King’s B
medium, while the remaining strains were grown in
YMB medium. In order to prepare cultures of microor-
ganisms in iron-rich media, 10 ml of the corresponding
liquid media was inoculated with 0.1 ml of the overnight
cultures and then incubated. The iron-deficient minimal
medium cultures were also prepared in this way. In
summary, 10 ml of RM-Fe liquid medium was inocu-
lated with 0.1 ml of the cultures that had been stressed
for 24 h under iron-deficient conditions (to remove the
presence of iron). Subsequently, the bacterial cultures
were incubated. All of these cultures were incubated
under mechanical shaking at 150 rev.ymin and a tem-
perature of 28–30 8C for 24 h. Subsequently, the cells
were pelleted by centrifugation at 10 000=g at 4 8C for
12 min, and then all bacterial suspensions were adjusted,
using sterile water, to an absorbance of 0.9 at 660 nm
wequivalent to approx. 109 colony-forming units (cfu)y
mlx.
2.3. Plant material
Medicago sativa (alfalfa) seeds of the Malone variety
were obtained from controlled agricultural field studies
conducted at New Mexico State University near Las
Cruces, New Mexico.
2.4. Seed inoculation and incubation
Cell suspensions (80 ml) of each bacterial strain
cultivated in iron-deficient and iron-rich media were
used to inoculate 25 alfalfa seeds separately on a Petri
dish (50=15 mm2). The seeds were air-dried at 26–28
8C for 60 min, and subsequently scattered on wet paper
towels (22=24 cm2). The wet paper towels containing
the inoculated seeds were then rolled up, placed into
plastic bags and kept in a vertical position to be
incubated at 28–30 8C in the dark. After 2 days, the
germinated seeds were then placed on the edge of new,
wet paper towels and were rolled up, and again kept in
a vertical position with the seedlings on top. The
seedlings were then incubated for 4 more days at 28–
30 8C with a photoperiod of 16 h. The basic experiment
had 37 treatments: 36 corresponding to the strains and
393
one to uninoculated control seeds. There were two
controls used, one with the presence of iron and one
without iron. Three replicate experiments were
performed.
2.5. Evaluation of seed germination and plant growth
After 24 h of incubation in the dark, the number of
germinated seeds was determined in each treatment.
The length of the roots and stems were measured after
6 days of incubation. A sample of 10 normal seedlings
from each treatment batch was selected and the roots
were separated from the shoots. Subsequently, the roots
and shoots were weighed, oven-dried at 70 8C for 24 h,
and then the dry weights were determined.
2.6. Statistical analysis
All the data were analyzed by analysis of variance
(ANOVA), and the treatment means were compared by
using the Tukey’s honestly significantly different (HSD)
multiple comparison test.
3. Results and discussion
3.1. Bacterial growth
The turbidity of bacterial cultures is an indication of
culture growth. In this research, the turbidity readings
of cultures developed in iron-rich media varied from
0.72 (strain Lc) to 1.5 (strains CP Mex. 46 and UAP
154), while the turbidity readings of cultures developed
in iron-deficient medium ranged from 0.7 (UAP 154)
to 1.2 (Sp 59). These results indicated that the strains
had the ability to express high affinity systems for iron
assimilation when they were exposed to iron-deficient
environments. Normally, cells become iron-limited in
the stationary phase of the growth cycle, which induces
the production of several chemical compounds. It was
noted before inoculation that six out of nine cultures of
Pseudomonas strains became green, and that cultures of
Rhizobium, such as CP Mex. 46, turned an amber color.
Previous research conducted with these microbial cells
demonstrated that a change in culture color is an
indication of high levels of accumulation of siderophores
in the growth media (Carrillo-Castaneda
˜ and Alvarado,
2000).
3.2. Seed germination
The effects of bacterial inoculation on alfalfa seed
germination are presented in Table 2. From this table, it
is evident that, in all cases, the seeds inoculated with
cells developed in the iron-deficient medium showed
higher percentage germination than those inoculated
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G. Carrillo-Castaneda et al. / Advances in Environmental Research 6 (2002) 391–399

Table 2 the other hand, the seeds inoculated with the same
Differential response of alfalfa seed germination to inoculation bacterial strains cultured in iron-deficient medium devel-
with bacterial cultures grown in rich and iron-deficient medium oped roots that varied in length from 55 (Tb) to 66 mm
(U). The mean root length of the non-inoculated seed-
Strain Germination (%)
lings was 54.4 mm. Statistical analysis corresponding
Culture medium to these data showed that the inoculated plantlets pro-
duced root lengths statistically different as compared
Rich RM-Fe
with the control plantlets (P-0.05). Under iron-rich
Pp 44 57 conditions, the best results were obtained with strains
Pf 49 66 A9, Pf and A7; however, under iron-deficient conditions,
A7 47 69 the best results were produced by strains U, CP Mex.
A9 52 56 46, Ls and UAP40. It is important to mention that
A9m 48 63
inoculation with bacterial cells grown under iron-defi-
Avm 49 60
Tb 59 65 cient conditions produced plantlets with more uniform
Ts 51 64 roots (range of 10 mm between minimum and maximum
U 53 65 length). The same strains grown in iron-rich medium
´ 19
CP Mex. 50 58 produced a more heterogeneous response (range of 22
´ 44
CP Mex. 58 60 mm)
´ 46
CP Mex. 52 64 Root biomass production of alfalfa plantlets was also
Lc 45 60 affected by the growth conditions of the strains used in
Ls 52 68 this study. Fig. 1b shows the root wet weight of the
Sp7 47 61 plantlets inoculated with the microbial cells cultured
Sp59 53 69
with and without iron. The root wet weight of seedlings
UAP40 53 64
UAP154 45 63 obtained from seeds inoculated with bacteria grown in
H2O (control) 55
iron-rich conditions ranged from 55 (A7) to 75 mg (CP
Mex. 44), while this parameter for seeds inoculated
RM-Fe, iron-deficient medium. with the bacteria grown in iron-deficient medium ranged
from 55 (Tb) to 70 mg (CP Mex. 46 and UAP40). The
with bacterial cultures grown in iron-rich medium (as root wet weight of the uninoculated seeds was 63 mg
compared to the uninoculated control seeds). The inoc- (see Fig. 1b, strain 19). The root dry weights are shown
ulation procedure with bacterial cells developed in iron- in Fig. 1c: for seeds inoculated with bacterial cells
deficient conditions significantly increased (P-0.05) cultivated in iron-rich medium, this varied from 3.2
the percentage germination over uninoculated seeds in (Ts) to 4.1 mg (AVm), and for seeds inoculated with
11 out of 18 strains. These increases ranged from 1% bacterial cells grown in iron-deficient medium, from 2.7
(A9) to 14% (A7, Sp 59). However, the inoculation (A9, A9m) to 3.3 mg (Lc, UAP40). The average root
with bacterial cells developed in iron-rich medium did dry weight of the uninoculated seeds was 3.2 mg. These
not cause a significant increase in the germination rate: differences were not statistically significant, but it was
only the strains Tb and CP Mex. 44 caused increases in evident that the microorganisms caused an increase in
percentage germination (4 and 1%, respectively). It is root dry weight when they were grown in an iron-rich
important to mention that in the germination stage the medium.
bacterial cells do not act on roots; however, the ready
availability of siderophore-like compounds biosynthe- 3.4. Determination of alfalfa stem growth after bacterial
sized by bacterial cells in the germination environment inoculation
may provide a delicate metal balance favorable for seed
germination. In this investigation, no signs of fungal Inoculation with some bacterial strains also affected
contamination or seed deterioration were detected. the alfalfa stem length, and shoot wet and dry weights.
Fig. 2a shows that the stem length of seeds inoculated
3.3. Determination of alfalfa root growth after bacterial with bacterial cells grown in iron-rich media varied
inoculation from 12 (Ls) to 14 mm (A9m), while that of seeds
inoculated with the bacterial strains grown in iron-
Fig. 1a shows the root length of alfalfa plantlets deficient medium varied from 13.0 (CP Mex. 19) to 17
developed from seeds inoculated with bacterial cultures mm (UAP40). The average stem length of the unino-
grown in iron-rich and iron-deficient media. In this culated seeds was 14.5 mm. ANOVA for these data
figure it is evident that seeds inoculated with microbial shows that the differences were statistically significant
cells grown in iron-rich medium produced roots lengths (P-0.05). Although the differences in stem wet and
that varied from 37.2 (UAP 154) to 59.3 mm (A9). On dry weight between uninoculated and inoculated seeds
 ˜
G. Carrillo-Castaneda et al. / Advances in Environmental Research 6 (2002) 391–399 395

Fig. 1. Influence of bacterial inoculation on: (a) alfalfa root length; (b) root wet weight; and (c) root dry weight. Bacterial strains
were cultured in iron-rich media and iron-deficient minimal medium (RM-Fe). Measurements were performed once the seedlings
had grown for 6 days. The No 19 strain corresponded to the uninoculated seeds. See Table 1 for strain description.
396 ˜
G. Carrillo-Castaneda et al. / Advances in Environmental Research 6 (2002) 391–399

Fig. 2. Responses to bacterial inoculation on: (a) alfalfa stem height; (b) stem wet weight; and (c) stem dry weight. Bacterial
strains were cultured in iron-rich media and iron-deficient minimal medium (RM-Fe). Measurements were performed once the
seedlings had grown for 6 days. The No 19 strain corresponded to the uninoculated seeds. See Table 1 for strain description.
 ˜
G. Carrillo-Castaneda et al. / Advances in Environmental Research 6 (2002) 391–399
Fig. 3. Comparison of the plant growth-promoting effects of bacterial stains on alfalfa seedlings. Bacterial strains were cultured in
iron-rich media and iron-deficient minimal medium (RM-Fe). Measurements were performed after 6 days of growth. The No 19
strain corresponded to the uninoculated seeds. See Table 1 for strain description.
were not statistically significant, it was possible to
observe that inoculation with some strains caused an
important increase in alfalfa stem weight (Fig. 2b,c).
3.5. Evaluation of the effect of culture media on bacte-
rial strains
Interesting results were obtained when examining the
effects of the growth-promoting activities of bacterial
inoculants depending on their culture condition. The U
strain promoted root elongation, but only when the
culture had been developed under iron-deficient minimal
medium conditions, whereas the A9 strain promoted
root elongation when it was cultured in iron-rich medi-
um (Fig. 1a). It is not completely understood what
microbial expressions determined these differences in
activity. Others have proposed different hypotheses to
explain these findings. Several investigators have
hypothesized that many types of biological activity
expressed by these strains may or may not be affected
by the media in which the bacterial cultures were grown.
Two of these are the production of antibiotics (Shanahan
et al., 1992) and cyanide (Flaishman et al., 1996),
which improve the phytosanitary conditions of the plant.
Another type of activity is the production of plant
hormones (Tien et al., 1979; Mahmoud et al., 1984;
Mordukhava et al., 1991), which induces root elonga-
tion, additional root hairs and lateral root formation
(Tien et al., 1979). Other investigators have proposed
that microorganisms synthesize siderophores in response
to iron starvation. These siderophores are capable of
binding other metal ions, such as aluminum, gallium,
chromium and copper (Emery, 1977). Therefore, sider-
ophores may facilitate the entry into the plant cells of
certain metal ions, such as iron (Neilands and Leong,
1986). This phenomenon can alleviate the iron deficien-
397
cy that causes the cessation of cell division in plant
cells (Abbott, 1972). On the other hand, plant growth-
promoting bacteria decrease the toxicity of some metals,
such as Cu, Ni and Fe, in the plant roots and stems,
because metals may be stored in the plant cells in a
non-toxic (chelated) form (Emery, 1977; Clarke et al.,
1987; Burd et al., 1998, 2000; Wallace et al., 1992). In
addition, siderophores increase the solubility of precip-
itated iron in soils and facilitate iron assimilation by the
plant (Bar-Ness et al., 1992; Yehuda et al., 1996). The
presence of iron-sequestering siderophores in the media
may also hinder colonization of hosts by pathogen
microorganisms, thereby suppressing diseases they may
cause. It has been documented that microorganisms
grown in iron-deficient minimal media develop a greater
capacity for biosynthesis, allowing them to produce a
wide range of metabolites necessary to grow (Neilands
and Leong, 1986; Carrillo-Castaneda˜ and Peralta, 1988).
In natural environments, microorganisms tolerate depri-
vation and adapt to utilizing organic nutrients excreted
by plant roots. These microorganisms may express genes
involved in nutrient acquisition, stress response and
secretion during early rhizosphere colonization, which
will help the plant resist aggressive environments (Rai-
ney, 1999)
Microbial effects became more significant when the
whole plant was taken into account. The seedlings
inoculated with the strains grown in iron-rich media
grew less, particularly strains of Azospirillum and Rhi-
zobium in comparison with Pseudomonas strains. The
largest seedlings were obtained with strain A9 (72 mm),
and smaller seedlings with strains UAP 154 (50 mm)
and Ls (57 mm). Only A9, CP Mex. 44, A9m, Pf and
Tb surpassed the non-inoculated seedling mean size (68
mm) (see Fig. 3, strain 19). The overall growth pro-
motion obtained with cultures developed in iron-defi-
398 ˜
G. Carrillo-Castaneda et al. / Advances in Environmental Research 6 (2002) 391–399
cient minimal medium was greater than that obtained
from inoculation with cultures grown in iron-rich media.
The largest mean seedling size was 81 mm for seeds
inoculated with strain U (Fig. 3), and the smallest was
for strain Pf (70 mm). These differences were statisti-
cally significant (P-0.05) as compared with growth of
the uninoculated seeds. It is important to take into
account that these differences have been observed in 6-
day-old seedlings. These promoting effects are currently
studied in developed plants and are usually related to
crop yields. It is hoped that results of this study may
reflect the behavior that will be observed in fully
developed inoculated plants. Previous studies have
shown that wheat seed inoculated with Azospirillum
manifested alterations in cell division of root tips and
the root elongation zone only 48 h after inoculation
(Bashan, 1986; Levanony and Bashan, 1989). Further-
more, several investigators have found that young plants
of several plant species inoculated with different Azos-
pirillum strains developed more rapidly compared with
uninoculated control plants (Bashan and Holguin, 1997;
Bashan et al., 1999; Carrillo-Garcia et al., 2000).
4. Conclusions
Results from this study have shown positive effects
on alfalfa plant growth in the early stages of develop-
ment due to the presence of bacterial cultures and their
expressions. Seed germination and the overall plantlet
growth promotion observed with cultures developed
under iron-deficient minimal medium conditions was
greater than that for seedlings inoculated with cells
grown in iron-rich media. The greatest root length was
obtained when seeds were inoculated with cells of strain
U cultured in iron-deficient minimal medium. These
seedlings also presented the greatest overall dimension.
Several strains have been shown to be more beneficial
to plant growth than others, particularly Pseudomonas
strains. This procedure is worthy for screening and
selecting plant growth-promoting bacteria, and we are
currently performing studies to determine the effects of
alfalfa seed inoculation with these microorganisms on
the ability of this plant to tolerate and uptake heavy
metal ions. Consequently, this study could have possible
application in the phytoremediation of heavy metal-
contaminated soils.
Acknowledgments
The authors acknowledge financial support from the
Center for Environmental Resource Management
(CERM) at the University of Texas at El Paso through
funding from the Office of Exploratory Research of the
US EPA (Cooperative Agreement CR-819849-01-04).
Dr Gardea-Torresdey would also like to acknowledge
the financial support of the National Institutes of Health
(Grant S06 GM8012-30).
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