Factors affecting the attachment of rhiz (2)
Factors a¡ecting the attachment of rhizospheric bacteria to
bean and soybean roots
Marta Albareda1, Marta Susana Dardanelli2, Carolina Sousa2, Manuel Meg´ıas2, Francisco Temprano1 &
Dulce N. Rodr´ıguez-Navarro1
1
CIFA-Las Torres-Tomejil (IFAPA), Alcala´ del R´ıo, Sevilla, Spain; and 2Departamento de Microbiolog´ıa y Parasitolog´ıa, Facultad de Farmacia, Universidad
de Sevilla, Sevilla, Spain
Correspondence: Dulce N. Rodr´ıguezNavarro, CIFA-Las Torres-Tomejil (IFAPA),
41200 Alcala´ del R´ıo, Sevilla, Spain. Tel.: 134
955 04 55 04; fax: 134 955 04 56 25; e-mail:
dulcenombre.rodriguez@juntadeandalucia.es
Received 3 January 2006; revised 9 March
2006; accepted 22 March 2006.
First published online 21 April 2006.
doi:10.1111/j.1574-6968.2006.00244.x
Editor: Yaacov Okon
Keywords
rhizobacteria; root-attachment; legumes.
Abstract
The plant rhizosphere is an important soil ecological environment for plant–
microorganism interactions, which include colonization by a variety of microorganisms in and around the roots that may result in symbiotic, endophytic,
associative, or parasitic relationships within the plant, depending on the type of
microorganisms, soil nutrient status, and soil environment. Rhizosphere competence may be attributable to the differences in the extent of bacterial attachment to
the root surface. We present results of the effect of various factors on the
attachment to bean (Phaseolus vulgaris) and soybean (Glycine max) roots of some
bacterial species of agronomic importance, such as Rhizobium tropici, Rhizobium
etli, Ensifer fredii (homotypic synonym Sinorhizobium fredii), and Azospirillum
brasilense; as well as the attachment capability of the plant growth promoting
rhizobacteria Pseudomonas fluorescens and Chryseobacterium balustinum. Additionally, we have studied various bacterial traits, such as autoaggregation and
flagella movements, which have been postulated to be important properties for
bacterial adhesion to surfaces. The lack of mutual incompatibility between
rhizobial strains and C. balustinum has been demonstrated in coinoculation assays.
Introduction
The plant rhizosphere is an important soil ecological
environment for plant–microorganism interactions, which
involves colonization by a variety of microorganisms in and
around the roots. The rhizosphere refers in general to the
portion of soil adjacent to the roots of living plants. It
supports a diverse and densely populated microbial community, and is subjected to chemical transformations caused
by the effect of root exudates and metabolites of microbial
degradation. The bacterial communities associated with this
microzone are thought to be determined by the quantity and
composition of root exudates that serve as substrate for
microbial growth. Root exudates can also selectively affect
the growth of bacteria and fungi that colonize the rhizosphere by serving as selective growth substrates for soil
microorganisms. These microbial associations may result in
endophytic, symbiotic, associative, or parasitic relationships
within the plant, depending on the type of microorganisms,
soil nutrient status, and soil environment (Parmar & Dadarwal, 1999). The best-known groups are symbiotic members of the family Rhizobiaceae, mycorrhizal fungi, and plant
growth promoting rhizobacteria (PGPR).
FEMS Microbiol Lett 259 (2006) 67–73
Attachment of Rhizobium/Bradyrhizobium to host roots is
supposedly the first requisite step for infection and nodulation. Various mechanisms and diverse surface molecules –
on both rhizobia and host plants – have been proposed to
mediate in this process. The first evidence of the role of
attachment as a specific step in the recognition between the
symbionts was presented by Bohlool & Schmidt (1974),
demonstrating a correlation between a strain’s ability to
bind soybean agglutinin (SBA) and to infect its legume host.
Further studies led to the evidence suggesting that attachment to soybean roots might be nonspecific and not involve
SBA-mediated adhesion (Meyer & Pueppke, 1980; Law et al.,
1982). Other hypotheses were formulated to describe physicochemical mechanisms of attachment that may be acting in
concert with, or instead of, SBA-mediated adhesion: anionic
repulsion between the bacterial and plant surfaces might
limit attachment; thus positive-charged compounds – such
as calcium – should stimulate adhesion, and hydrophobic
interactions could also be important in the plant–microorganism interaction. Other studies have demonstrated an
active adsorption to roots by polymeric fibrillar bridges
or via protein bridges. Factors affecting the attachment
c 2006 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. No claim to original Spanish government works
68
between the symbionts have also been the subject of debate,
including culture age, bacterial and root pretreatment conditions, bacterial chemotaxis and motility, presence and
extension of bacterial fimbriation (Vesper & Bauer, 1986),
and bacterial surface polysaccharides (EPS and LPS).
In a framework of sustainable agriculture, microbial soil
diversity is promoted by certain practices that lead to better
nutrient cycling, disease suppression, and nitrogen fixation.
One of these is the introduction of beneficial bacteria into
soil, through the use of microbial inoculants (biofertilizers)
based on single species (e.g. Rhizobium) inoculants or on
several PGPRs species. Biofertilizer is a recently coined term
whose exact definition is still unclear, but which most
commonly refers to the use of soil microorganisms to
increase the availability and uptake of mineral nutrients for
plants. The experiments carried out in this work are in the
context of a national project whose main goal is to optimize
the symbiotic association between soybean/Ensifer fredii and
bean/Rhizobium spp. using mixed inoculants. In this framework, it has to be provided that there are no problems of
mutual exclusion, displacement, or competence between the
inoculant strains. Before these beneficial biofertilizers are
formulated and released into the environment, preliminary
studies are needed on the first stages of root colonization
(attachment) and on absence of mutual incompatibility.
The aim of this work has been the study of some factors
that may affect bacterial attachment to legume roots: (i)
presence of salt (50 mM of NaCl), (ii) culture age, (iii) size of
the inoculum, (iv) pH, and (v) presence of other rhizospheric bacteria. Additionally, some bacterial traits related to
surface adhesion have been investigated, as well as the ability
of coinoculated bacteria to grow in root exudates. Our data
suggest that the optimum attachment is dependent of the
bacteria added. The presence of moderate salt concentration
in the plant rooting medium or acidification of the attachment buffer are factors that might affect bacterial adhesion.
The culture age of the inoculated bacteria was found to be
important for adhesion; thus, attachment of stationary
phase cells is greater than that of exponential phase cells.
The presence of Chryseobacterium balustinum (up to
105 CFU mL 1) affected adhesion of Ensifer fredii strains to
soybean roots. Finally, the degree of rhizobacterial root
attachment seems to be independent of certain bacterial
surface properties, such as autoaggregation and flagella
movement, and of the legume–host species.
Materials and methods
Bacterial strains and growth media
The bacterial strains used in this work were Pseudomonas
fluorescens strain WCS417r (Pieterse et al., 1996), Azospirillum brasilense strain Sp7 (Tarrand et al., 1978), Chryseo2006 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. No claim to original Spanish government works
c
M. Albareda et al.
bacterium balustinum strain Aur9 (Gutie´ rrez-Ma˜nero et al.,
2003), Rhizobium tropici strain CIAT899 (Mart´ınez-Romero
et al., 1991), Rhizobium etli strain ISP42 (Rodr´ıguezNavarro et al., 2000), E. fredii strain HH103 (Dowdle &
Bohlool, 1985) and E. fredii strain SMH12 (Cleyet-Marel,
1987).
All rhizobial and Pseudomonas strains were grown in
B medium (Spaink et al., 1992), Azospirillum brasilense
was grown in NFb (D¨obereiner & Baldani, 1979), and C.
balustinum was grown in TY (Beringer, 1974), with shaking
at 28 1C, until stationary (OD600 nm 4 1.0) or late exponential (OD600 nm = 0.5) phase. For studies under saline conditions, bacterial media were supplemented with 50 mM of
NaCl.
Plant species and growth conditions
We used 7-day-old roots of bean and soybean for attachment studies. Surface disinfections of bean (cv. BBL) and
soybean (cv. Osumi) seeds, germination, and growth on
nitrogen-free medium have been described elsewhere (Camacho et al., 2002). Germinated seeds were aseptically
transferred to the sterile growth system. This system was
designed to allow the growth of seedlings in hydroponic
conditions, and consists of a stainless-steel sieve coupled to a
glass reservoir of c. 200 mL (Rigaud & Puppo, 1975). N-free
plant nutrient solution (1/2 strength, pH 6.8) was used for
plant growth. Plants were maintained in a growth chamber
at 28 1C (14 h) and 18 1C (10 h) under a photosynthetically
active radiation of 124 mE m 2 s 1 provided by fluorescent
tubes for 7 days.
Roots were then removed; portions of c. 1 cm were cut off,
and collected in 25 mM phosphate buffer (pH 7.5). When
saline conditions were imposed, 50 mM of NaCl was added
to the nutrient solution. For studies with preinoculated
roots, plantlets of bean or soybean were inoculated with C.
balustinum strain Aur9 to a final density of 106 CFU mL 1,
15 h or 48 h, respectively, before rhizobial strains were
inoculated.
Bacterial attachment assay
Five root pieces (1 cm in length) were aseptically transferred
to an Eppendorf tube with 1 mL 25 mM phosphate buffer
(pH 7.5) (or pH 5.5 when acidic conditions were imposed).
Bacterial cells were harvested by centrifugation at 8064 g for
5 min, washed twice with 25 mM phosphate buffer (pH 7.5),
resuspended in 100 mL of the same buffer, and then added to
roots. Inoculated roots were incubated at room temperature
for 1 h in a swing shaker, then washed eight times with
phosphate buffer, and transferred to a sterile mortar and
pestle and homogenized. Serial decimal dilutions were
performed, and plated on TY-agar media for root-bound
bacteria enumeration.
FEMS Microbiol Lett 259 (2006) 67–73
69
Attachment of rhizospheric bacteria to bean and soybean roots
Table 1. Attachment of different plant growth promoting rhizobacteria to legume roots under control and salty conditions
Rhizobacterial species
Plant conditions
Phaseolus vulgaris
Control
Salt
Glycine max
Control
Salt
Pseudomonas fluorescens (WCS417r)
Azospirillum brasilense (Sp7)
Chryseobacterium balustinum (Aur9)
5.73 0.38a
5.67 0.44a
5.02 0.26a
5.39 0.33a
4.93 0.40c
5.87 0.11b
6.22 0.30a
6.53 0.34a
5.44 0.43a
4.82 0.70a
6.22 0.16ab
6.75 0.17a
Data represent mean log10 SD values of attached bacteria, from at least two independent assays. Significant differences (P o 0.10) between values
within a column are indicated by different letters.
Salt concentration: 50 mM NaCl.
Table 2. Attachment of rhizobial strains to legume roots under control and salty conditions
Rhizobial strain
Rhizobium tropici (CIAT899)
Plant conditions
Control
Salt
Rhizobium etli (ISP42)
Ensifer fredii (SMH12)
Phaseolus vulgaris
6.34 0.08a
5.92 0.27a
Ensifer fredii (HH103)
Glycine max
5.43 0.16a
4.88 0.21b
5.24 0.07a
3.64 0.16b
5.13 0.34a
3.46 0.06b
Data represent mean log10 SD values of attached bacteria, from at least two independent assays. Significant differences (P o 0.10) between values
within a column are indicated by different letters.
Salt concentration: 50 mM NaCl.
To determine rhizobial attachment based on inoculum
size, saturated cultures of CIAT899 and SMH12 strains were
obtained (109 cells mL 1), and decimal dilutions were inoculated on bean or soybean roots, respectively. Then the
adhesiveness assay was performed as above.
Autoaggregation assay
Autoaggregation assays were performed according to Kos
et al. (2003), with minor modifications. Rhizobial species
were grown in yeast-mannitol broth, and Pseudomonas and
Chryseobacterium in liquid TY, at 25 1C to give cultures of c.
108 CFU mL 1. The cells were harvested by centrifugation at
5000 g for 15 min, washed twice, and resuspended in phosphate-buffered saline (PBS). Cell suspensions were mixed by
vortexing, and autoaggregation was determined during 24 h
of static incubation at room temperatures. At intervals of
4 h, 0.1 mL of the upper layer of the bacterial suspension was
transferred to another tube with 3.9 mL of PBS, and the
absorbance (A) was measured at 600 nm. Autoaggregation
percentage is expressed as 1 (At/A0) 100, where At
represents the absorbance at different sampling times, and
A0 the absorbance at t = 0.
Motility assays
Swimming and swarming motilities were analysed according
to the methods described by De´ ziel et al. (2001). Ensifer
fredii strain HH103 was grown on soft yeast-mannitol agar.
FEMS Microbiol Lett 259 (2006) 67–73
Data analysis
All the experiments were carried out in duplicate. One-way
ANOVA was used to analyze the results of the inoculum size
assay, using Statistix 7.0. When the analysis of variance
showed significant treatment effect, the least significant
differences test (LSD, P o 0.05) was applied to make comparisons between the means.
Results
Attachment of different rhizobacteria to legume
roots
All the strains used in this work have been tested for their
capacity to attach to both bean and soybean roots, under
control and saline conditions (Tables 1 and 2). When
moderately saline conditions were tested, plantlets and
bacteria were grown in the presence of 50 mM NaCl.
Attachment of the PGPR strains Pseudomonas fluorescens,
A. brasilense, and C. balustinum to both legume roots was,
in general, not affected in the presence of moderate salt
concentration, but C. balustinum showed a better attachment capacity under saline conditions; moreover, P. fluorescens WCS417r and C. balustinum Aur9 cells did show a
significant (P o 0.10) preferential adhesion to soybean
roots.
Adhesion of rhizobial strains to specific (E. fredii strains/
soybean and R. tropici, R. etli/bean) and nonspecific legume
c 2006 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. No claim to original Spanish government works
70
M. Albareda et al.
roots was studied (Tables 2 and 3). Ensifer fredii strains
SMH12 and HH103 showed a low degree of root adhesion,
in comparison with the other rhizobial and PGPR species;
moreover, the attachment to soybean roots grown under
saline conditions significantly (P o 0.10) declined in relation to control roots. Of the rhizobial bean-nodulating
species, R. tropici strain CIAT899 showed a higher degree of
root attachment than did R. etli strain ISP42; however, there
was no differences between species in the attachment to
bean or soybean roots (nonspecific legume) (data not
shown). The presence of salt significantly reduced the
adhesion of R. etli to bean roots (Table 2).
Effect of culture age, inoculum size, and pH on
root attachment
The effect of culture age of the inoculant strains on root
attachment was investigated with specific rhizobia/legume
combinations and with C. balustinum Aur9 on both legume
species (Table 3). In general, bacterial cells at stationary phase
showed a higher capacity to adhere to roots, but in only two
of six cases studied (C. balustinum/Phaseolus and R. tropici/
Phaseolus) was the rate of adhesion significantly higher
(P o 0.10). The effect of the size of the inoculum of R. tropici
CIAT899 and E. fredii SMH12 strains on the attachment to
bean and soybean roots, respectively, was tested (Table 4). In
the case of SMH12, a significantly higher number of rhizobia
attached to roots when the applied inoculum was in the range
of 108–109 CFU mL 1 compared with diluted inocula
(107 CFU mL 1). More-dilute inocula (105–106 CFU mL 1)
gave levels of attachment below quantifiable limits (BQL). In
contrast, saturated cultures of CIAT899 led to significantly
fewer attached cells, with 105–106 CFU mL 1 giving a maximum level of root adhesion.
The effect of pH on the attachment of three rhizobacteria
to the two legumes was studied. Both specific and nonspecific rhizobial strains and C. balustinum were selected for
these studies. The pH of the incubation buffer had a slight
effect on the bacterial attachment to legume roots. At acidic
pH (5.5), there was in general a lower ratio of adhesion to
roots, but with some exceptions (SMH12/Phaseolus and
Aur9/Glycine) (data not shown).
Bacteria traits associated with adhesion
In order to elucidate whether some bacterial traits might be
responsible for the low degree of rhizobial root attachment,
we performed studies of autoaggregation and flagella-associated movement, as bacterial properties linked to surface
adhesion. There was no correlation between root attachment and the autoaggregation ability of the studied bacteria.
The highest percentage of autoaggregation was shown by E.
fredii strains SMH12 (47–62%) and HH103 (65–88%) after
24 h of incubation; on the other hand, these strains had
shown the lowest degree of attachment. For the other
bacterial species, the percentage of autoaggregation varied
from 30% to 40%.
All the studied bacteria showed both modes of flagellaassociated motilities. Swimming motility (owing to the
presence of polar flagella) was determined on plates (0.3%
agar). In the rhizobial strains, the greatest motility was
observed with R. etli strain ISP42 (90 mm) and E. fredii
strain HH103 (80 mm) cells. The rhizobacteria P. fluorescens
showed the greatest movement (75 mm), and C. balustinum
showed a dispersal growth from the point of inoculation
without clear growth rings. Swarming motility (owing to the
presence of lateral flagella) was determined on 0.5% agar
plates. All the strains showed a very uniform swarming
ability, with growth rings ranging from 10 to 30 mm.
Attachment of rhizobial strains to preinoculated
roots with C. balustinum
The effect of the presence of C. balustinum (Aur9) in the
rooting system of bean and soybean plantlets was investigated, before the rhizobial attachment assay using specific
rhizobia/legume combinations, under control and saline
conditions [Tables 5(a) and (b)]. Strain Aur9 was added to
the plant rooting medium 14 h (bean) or 48 h (soybean)
before the attachment assay. The presence of Aur9 (ranging
Table 3. Effect of bacterial growth phase on the attachment of rhizobacterial strains
Bacterial strains
Growth phase
Phaseolus vulgaris
Stationary
Exponential
Glycine max
Stationary
Exponential
Chryseobacterium balustinum
(Aur9)
Rhizobium tropici
(CIAT899)
Rhizobium etli
(ISP42)
Ensifer fredii
(SMH12)
Ensifer fredii
(HH103)
6.23 0.01a
5.31 0.20b
5.93 0.27a
3.82 0.58b
5.59 0.30a
4.96 0.49a
nt
nt
nt
nt
6.37 0.12a
5.11 0.94a
nt
nt
nt
nt
5.86 0.22a
5.21 0.42a
5.47 0.01a
4.71 0.30a
Data represent mean log10 SD values of attached bacteria, from at least two independent assays. Significant differences (P o 0.10) between values
within a column are indicated by different letters.
nt, not tested.
c 2006 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. No claim to original Spanish government works
FEMS Microbiol Lett 259 (2006) 67–73
71
Attachment of rhizospheric bacteria to bean and soybean roots
103–104 CFU mL 1) did not impair the capacity of beannodulating species to attach to bean roots. In the case of
E. fredii strains, the presence of Aur9 (104–106 CFU mL 1)
lessened their capacity of attachment to soybean roots;
however, under saline conditions, the adhesion of the
SMH12 strain to preinduced soybean roots was significantly
better than to control roots. The adhesion of the CIAT899
strain to preinduced bean roots, under saline conditions,
was also better. In the case of the R. etli ISP42 strain, there
was no variation in its attachment capacity under the
different assay conditions.
Discussion
PGPRs have drawn much attention in recent years because
of their contribution to the biological control of plant
pathogens and the improvement of plant growth. Inoculation of plants with ‘dual’ microbial inoculants, or even a
consortium of them, is becoming more important in a
framework of sustainable agriculture for the advantage their
beneficial effects afford, providing there is no competition
Table 4. Effect of bacterial inoculum size on the attachment of rhizobial
strains to specific legume–host roots
Inoculum (Lg CFU mL 1)
Strains
9
8
7
6
5
SMH12
CIAT899
52.4b
88.8c
84.3a
260.8b
11.1c
245.8b
BQL
925.5a
BQL
803.8a
Data are means of two replicates. Significant differences (P o 0.05)
between values within each row are indicated by different letters.
BQL, below quantifiable limits.
between inoculants. However, the practical use of these
beneficial bacteria sometimes fails because of their inability
to colonize the rhizosphere or the rhizoplane of inoculated
plants. Colonization of roots by inoculant strains thus
appears to be a critical step in the interaction between
beneficial bacteria and host plants. Attachment of rhizobia
to host roots seems to be the first requisite step in infection
and nodulation (Bohlool & Schmidt, 1974), and is considered one of the physiologically important characteristics in
determining competitive ability among rhizobial strains
(Smith & Wollum, 1991).
Root colonization is defined as a complex phenomenon
depending upon several biotic and abiotic factors. In this
work, we have defined a hydroponic system to grow plants
under bacteriologically controlled conditions, allowing
changes in the rooting media (saline or control conditions),
the controlled addition of bacterial inoculants, or pH
changes. The attachment assay used in this work is similar
to that of Dardanelli et al. (2003), and the values for
bacterial attachment presented here can be considered those
of tightly attached bacteria. Chabot et al. (1996) have
described rhizobial strains that were better colonizers of
maize and lettuce roots than were other PGPR bacteria; our
results, however, indicated that the studied PGPRs are in
general better colonizers of the two legumes than are the
specific rhizobial species, except Rhizobium tropici/bean
roots. An attempt was made to correlate the low degree of
rhizobial attachment with physicochemical characteristics of
the cell surface by measuring the autoaggregation of the
studied rhizobacteria, as well as the flagella-associated
motilities. There is a relationship between the bacterial
ability for autoaggregation and that of adhesion to different
Table 5. Attachment of (a) bean-nodulating and (b) soybean-nodulating strains to non- or preinoculated roots with Chryseobacterium balustinum
strain Aur9
Rhizobial strains
Presence of C. balustinum
(a) Bean-nodulating strains
Non
1 Aur9
Plant growth conditions
Rhizobium tropici CIAT899
Rhizobium etli ISP42
Control
Salt
Control
Salt
6.34 0.08ab
5.92 0.27b
6.33 0.10ab
6.56 0.01a
5.42 0.11a
4.88 0.21a
4.78 0.34a
5.40 0.42a
Rhizobial strains
Presence of C. balustinum
(b) Soybean-nodulating strains
Non
1Aur9
Plant growth conditions
Ensifer fredii HH103
Ensifer fredii SMH12
Control
Salt
Control
Salt
5.13 0.34a
3.45 0.06b
4.23 0.26b
nt
5.24 0.08a
3.64 0.16d
5.03 0.02b
4.18 0.02c
Data represent mean log10 SD values of attached bacteria, from at least two independent assays. Significant differences (P o 0.10) between values
within a column are indicated by different letters.
nt, not tested.
FEMS Microbiol Lett 259 (2006) 67–73
c 2006 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. No claim to original Spanish government works
72
surfaces (Zaady & Okon, 1990; Kos et al., 2003). Our data
agree with those of Zaady & Okon, as they have shown that
Azospirillum cell treatments leading to strong inhibition of
aggregation culminate in the greatest adsorption to maize
roots. In our case, the highest autoaggregation percentage
was shown by E. fredii strains, which showed the lowest
degree of root attachment. However, our results do not agree
with those obtained by Kos et al. (2003) using Lactobacillus
strains. It has been suggested that anionic repulsions
between root and rhizobia may explain why only a small
proportion of a rhizobial population attaches to legume
roots (Vesper & Bauer, 1985; Caetano-Anolles & Favelukes,
1986). In addition, bacterial cell movements because of
flagella have been linked to adherence of some Gramnegative gastrointestinal bacteria to epithelial cells in vitro
and to other inert surfaces. We have determined the modes
of flagella-associated motilities, owing to polar and lateral
flagella, and found no defective swimming or swarming
motilities that might explain the differences in root attachment capacity of the studied bacteria.
The level of rhizobial adhesion to the specific or nonspecific host legume roots was similar for all studied species,
although bean-nodulating rhizobia are better colonizers
than soybean-E. fredii microsymbionts. Nevertheless, in our
study the proportion of attached cells with respect to
inoculated levels is much lower than that reported in other
studies (Vesper & Bauer, 1985, 1986). These results reinforce
the concept of lack of specificity of host legume exudates for
their homologous rhizobial microsymbionts, as has been
largely demonstrated (Pueppke, 1984; Vesper & Bauer, 1985;
Dolhem-Biremon et al., 1993). A nonspecific mechanism,
independent of the symbiotic properties, must contribute to
root attachment of rhizobia, as the same colonization level
has been reported for P. fluorescens and Sinorhizobium
meliloti in alfalfa roots (Villacieros et al., 2003). However, a
certain specificity of plant exudates to their bacterial isolates
have also been proposed (Mandimba et al., 1986; BacilioJime´ nez et al., 2003).
It has been reported that culture age affects the extent of
bradyrhizobial attachment to soybean roots, and that it is
strain-dependent (Smith & Wollum, 1991). We have also
found that culture age has a distinct effect on root attachment of certain strains, and there was a general and positive
tendency for a greater attachment level in stationary cells.
Although bacterial cultures were twice washed and centrifuged with buffer, so that extracellular polymeric substances
should have been removed, when cells enter the stationary
phase of growth, other morphological and biochemical
changes occur that might modify the degree of attachment.
Moreover, acidification (pH 5.5) of the attachment buffer
did not greatly affect the adhesion of studied bacteria in
comparison with control buffer (pH 7.5). Possible modifications of the bacterial cell surface during 1 h of incubation
2006 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. No claim to original Spanish government works
c
M. Albareda et al.
might account for the slight differences observed, as changes
in root surface are probably negligible; in fact, the final pH
of the rooting medium usually decreases after 1 week of root
development. Using two organic buffering systems, Smith &
Wollum (1993) found an optimum pH range (6.0–6.5) for
the attachment of Bradyrhizobium japonicum USDA110 to
soybean roots. By contrast, root surface modifications
occurring when 50 mM NaCl was added to the rooting
system might be strong grounds for some differences in
bacterial root attachment under saline conditions.
Root attachment was dose-dependent in the case of E.
fredii SMH12 strain, and optimum adhesion was found at
a concentration of 108–109 CFU mL 1, in agreement with
other results (Jjemba & Alexander, 1999). However, in the
case of R. tropici CIAT899, a large inoculum gave poor
attachment to bean roots. Differences in the optimum
attachment of these strains with varying inoculum size
might be attributable to differential bacterial traits rather
than to a limitation of binding sites in these root-segment
assays, as most of the attachment trials have been performed
with saturated bacterial inocula or in the presence of other
bacteria (preinoculated C. balustinum roots), and small
percentages of rhizobial populations were consistently
found to adhere firmly to roots.
The presence of C. balustinum Aur9 strain in the rooting
media 15–20 h before rhizobial-attachment assays did not
affect the adhesion of bean-nodulating species, but did
lessen the attachment of E. fredii strains; perhaps the higher
level of Aur9 in the soybean rooting media was the reason
for this difference.
The study has demonstrated that the coinoculation of
Aur9 and rhizobial strains provides the same growth capacity in root exudates as that of single inocula, but further
studies of proper root colonization dynamics, using whole
root systems, are necessary to reveal the actual binding
points on the roots, and to demonstrate persistence of the
studied strains in the rhizosphere.
Acknowledgements
´ y
This research was founded by the Ministerio de Educacion
Ciencia (Spain). Project: AGL2002-04188-CO6.
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c 2006 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. No claim to original Spanish government works
bean and soybean roots
Marta Albareda1, Marta Susana Dardanelli2, Carolina Sousa2, Manuel Meg´ıas2, Francisco Temprano1 &
Dulce N. Rodr´ıguez-Navarro1
1
CIFA-Las Torres-Tomejil (IFAPA), Alcala´ del R´ıo, Sevilla, Spain; and 2Departamento de Microbiolog´ıa y Parasitolog´ıa, Facultad de Farmacia, Universidad
de Sevilla, Sevilla, Spain
Correspondence: Dulce N. Rodr´ıguezNavarro, CIFA-Las Torres-Tomejil (IFAPA),
41200 Alcala´ del R´ıo, Sevilla, Spain. Tel.: 134
955 04 55 04; fax: 134 955 04 56 25; e-mail:
dulcenombre.rodriguez@juntadeandalucia.es
Received 3 January 2006; revised 9 March
2006; accepted 22 March 2006.
First published online 21 April 2006.
doi:10.1111/j.1574-6968.2006.00244.x
Editor: Yaacov Okon
Keywords
rhizobacteria; root-attachment; legumes.
Abstract
The plant rhizosphere is an important soil ecological environment for plant–
microorganism interactions, which include colonization by a variety of microorganisms in and around the roots that may result in symbiotic, endophytic,
associative, or parasitic relationships within the plant, depending on the type of
microorganisms, soil nutrient status, and soil environment. Rhizosphere competence may be attributable to the differences in the extent of bacterial attachment to
the root surface. We present results of the effect of various factors on the
attachment to bean (Phaseolus vulgaris) and soybean (Glycine max) roots of some
bacterial species of agronomic importance, such as Rhizobium tropici, Rhizobium
etli, Ensifer fredii (homotypic synonym Sinorhizobium fredii), and Azospirillum
brasilense; as well as the attachment capability of the plant growth promoting
rhizobacteria Pseudomonas fluorescens and Chryseobacterium balustinum. Additionally, we have studied various bacterial traits, such as autoaggregation and
flagella movements, which have been postulated to be important properties for
bacterial adhesion to surfaces. The lack of mutual incompatibility between
rhizobial strains and C. balustinum has been demonstrated in coinoculation assays.
Introduction
The plant rhizosphere is an important soil ecological
environment for plant–microorganism interactions, which
involves colonization by a variety of microorganisms in and
around the roots. The rhizosphere refers in general to the
portion of soil adjacent to the roots of living plants. It
supports a diverse and densely populated microbial community, and is subjected to chemical transformations caused
by the effect of root exudates and metabolites of microbial
degradation. The bacterial communities associated with this
microzone are thought to be determined by the quantity and
composition of root exudates that serve as substrate for
microbial growth. Root exudates can also selectively affect
the growth of bacteria and fungi that colonize the rhizosphere by serving as selective growth substrates for soil
microorganisms. These microbial associations may result in
endophytic, symbiotic, associative, or parasitic relationships
within the plant, depending on the type of microorganisms,
soil nutrient status, and soil environment (Parmar & Dadarwal, 1999). The best-known groups are symbiotic members of the family Rhizobiaceae, mycorrhizal fungi, and plant
growth promoting rhizobacteria (PGPR).
FEMS Microbiol Lett 259 (2006) 67–73
Attachment of Rhizobium/Bradyrhizobium to host roots is
supposedly the first requisite step for infection and nodulation. Various mechanisms and diverse surface molecules –
on both rhizobia and host plants – have been proposed to
mediate in this process. The first evidence of the role of
attachment as a specific step in the recognition between the
symbionts was presented by Bohlool & Schmidt (1974),
demonstrating a correlation between a strain’s ability to
bind soybean agglutinin (SBA) and to infect its legume host.
Further studies led to the evidence suggesting that attachment to soybean roots might be nonspecific and not involve
SBA-mediated adhesion (Meyer & Pueppke, 1980; Law et al.,
1982). Other hypotheses were formulated to describe physicochemical mechanisms of attachment that may be acting in
concert with, or instead of, SBA-mediated adhesion: anionic
repulsion between the bacterial and plant surfaces might
limit attachment; thus positive-charged compounds – such
as calcium – should stimulate adhesion, and hydrophobic
interactions could also be important in the plant–microorganism interaction. Other studies have demonstrated an
active adsorption to roots by polymeric fibrillar bridges
or via protein bridges. Factors affecting the attachment
c 2006 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. No claim to original Spanish government works
68
between the symbionts have also been the subject of debate,
including culture age, bacterial and root pretreatment conditions, bacterial chemotaxis and motility, presence and
extension of bacterial fimbriation (Vesper & Bauer, 1986),
and bacterial surface polysaccharides (EPS and LPS).
In a framework of sustainable agriculture, microbial soil
diversity is promoted by certain practices that lead to better
nutrient cycling, disease suppression, and nitrogen fixation.
One of these is the introduction of beneficial bacteria into
soil, through the use of microbial inoculants (biofertilizers)
based on single species (e.g. Rhizobium) inoculants or on
several PGPRs species. Biofertilizer is a recently coined term
whose exact definition is still unclear, but which most
commonly refers to the use of soil microorganisms to
increase the availability and uptake of mineral nutrients for
plants. The experiments carried out in this work are in the
context of a national project whose main goal is to optimize
the symbiotic association between soybean/Ensifer fredii and
bean/Rhizobium spp. using mixed inoculants. In this framework, it has to be provided that there are no problems of
mutual exclusion, displacement, or competence between the
inoculant strains. Before these beneficial biofertilizers are
formulated and released into the environment, preliminary
studies are needed on the first stages of root colonization
(attachment) and on absence of mutual incompatibility.
The aim of this work has been the study of some factors
that may affect bacterial attachment to legume roots: (i)
presence of salt (50 mM of NaCl), (ii) culture age, (iii) size of
the inoculum, (iv) pH, and (v) presence of other rhizospheric bacteria. Additionally, some bacterial traits related to
surface adhesion have been investigated, as well as the ability
of coinoculated bacteria to grow in root exudates. Our data
suggest that the optimum attachment is dependent of the
bacteria added. The presence of moderate salt concentration
in the plant rooting medium or acidification of the attachment buffer are factors that might affect bacterial adhesion.
The culture age of the inoculated bacteria was found to be
important for adhesion; thus, attachment of stationary
phase cells is greater than that of exponential phase cells.
The presence of Chryseobacterium balustinum (up to
105 CFU mL 1) affected adhesion of Ensifer fredii strains to
soybean roots. Finally, the degree of rhizobacterial root
attachment seems to be independent of certain bacterial
surface properties, such as autoaggregation and flagella
movement, and of the legume–host species.
Materials and methods
Bacterial strains and growth media
The bacterial strains used in this work were Pseudomonas
fluorescens strain WCS417r (Pieterse et al., 1996), Azospirillum brasilense strain Sp7 (Tarrand et al., 1978), Chryseo2006 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. No claim to original Spanish government works
c
M. Albareda et al.
bacterium balustinum strain Aur9 (Gutie´ rrez-Ma˜nero et al.,
2003), Rhizobium tropici strain CIAT899 (Mart´ınez-Romero
et al., 1991), Rhizobium etli strain ISP42 (Rodr´ıguezNavarro et al., 2000), E. fredii strain HH103 (Dowdle &
Bohlool, 1985) and E. fredii strain SMH12 (Cleyet-Marel,
1987).
All rhizobial and Pseudomonas strains were grown in
B medium (Spaink et al., 1992), Azospirillum brasilense
was grown in NFb (D¨obereiner & Baldani, 1979), and C.
balustinum was grown in TY (Beringer, 1974), with shaking
at 28 1C, until stationary (OD600 nm 4 1.0) or late exponential (OD600 nm = 0.5) phase. For studies under saline conditions, bacterial media were supplemented with 50 mM of
NaCl.
Plant species and growth conditions
We used 7-day-old roots of bean and soybean for attachment studies. Surface disinfections of bean (cv. BBL) and
soybean (cv. Osumi) seeds, germination, and growth on
nitrogen-free medium have been described elsewhere (Camacho et al., 2002). Germinated seeds were aseptically
transferred to the sterile growth system. This system was
designed to allow the growth of seedlings in hydroponic
conditions, and consists of a stainless-steel sieve coupled to a
glass reservoir of c. 200 mL (Rigaud & Puppo, 1975). N-free
plant nutrient solution (1/2 strength, pH 6.8) was used for
plant growth. Plants were maintained in a growth chamber
at 28 1C (14 h) and 18 1C (10 h) under a photosynthetically
active radiation of 124 mE m 2 s 1 provided by fluorescent
tubes for 7 days.
Roots were then removed; portions of c. 1 cm were cut off,
and collected in 25 mM phosphate buffer (pH 7.5). When
saline conditions were imposed, 50 mM of NaCl was added
to the nutrient solution. For studies with preinoculated
roots, plantlets of bean or soybean were inoculated with C.
balustinum strain Aur9 to a final density of 106 CFU mL 1,
15 h or 48 h, respectively, before rhizobial strains were
inoculated.
Bacterial attachment assay
Five root pieces (1 cm in length) were aseptically transferred
to an Eppendorf tube with 1 mL 25 mM phosphate buffer
(pH 7.5) (or pH 5.5 when acidic conditions were imposed).
Bacterial cells were harvested by centrifugation at 8064 g for
5 min, washed twice with 25 mM phosphate buffer (pH 7.5),
resuspended in 100 mL of the same buffer, and then added to
roots. Inoculated roots were incubated at room temperature
for 1 h in a swing shaker, then washed eight times with
phosphate buffer, and transferred to a sterile mortar and
pestle and homogenized. Serial decimal dilutions were
performed, and plated on TY-agar media for root-bound
bacteria enumeration.
FEMS Microbiol Lett 259 (2006) 67–73
69
Attachment of rhizospheric bacteria to bean and soybean roots
Table 1. Attachment of different plant growth promoting rhizobacteria to legume roots under control and salty conditions
Rhizobacterial species
Plant conditions
Phaseolus vulgaris
Control
Salt
Glycine max
Control
Salt
Pseudomonas fluorescens (WCS417r)
Azospirillum brasilense (Sp7)
Chryseobacterium balustinum (Aur9)
5.73 0.38a
5.67 0.44a
5.02 0.26a
5.39 0.33a
4.93 0.40c
5.87 0.11b
6.22 0.30a
6.53 0.34a
5.44 0.43a
4.82 0.70a
6.22 0.16ab
6.75 0.17a
Data represent mean log10 SD values of attached bacteria, from at least two independent assays. Significant differences (P o 0.10) between values
within a column are indicated by different letters.
Salt concentration: 50 mM NaCl.
Table 2. Attachment of rhizobial strains to legume roots under control and salty conditions
Rhizobial strain
Rhizobium tropici (CIAT899)
Plant conditions
Control
Salt
Rhizobium etli (ISP42)
Ensifer fredii (SMH12)
Phaseolus vulgaris
6.34 0.08a
5.92 0.27a
Ensifer fredii (HH103)
Glycine max
5.43 0.16a
4.88 0.21b
5.24 0.07a
3.64 0.16b
5.13 0.34a
3.46 0.06b
Data represent mean log10 SD values of attached bacteria, from at least two independent assays. Significant differences (P o 0.10) between values
within a column are indicated by different letters.
Salt concentration: 50 mM NaCl.
To determine rhizobial attachment based on inoculum
size, saturated cultures of CIAT899 and SMH12 strains were
obtained (109 cells mL 1), and decimal dilutions were inoculated on bean or soybean roots, respectively. Then the
adhesiveness assay was performed as above.
Autoaggregation assay
Autoaggregation assays were performed according to Kos
et al. (2003), with minor modifications. Rhizobial species
were grown in yeast-mannitol broth, and Pseudomonas and
Chryseobacterium in liquid TY, at 25 1C to give cultures of c.
108 CFU mL 1. The cells were harvested by centrifugation at
5000 g for 15 min, washed twice, and resuspended in phosphate-buffered saline (PBS). Cell suspensions were mixed by
vortexing, and autoaggregation was determined during 24 h
of static incubation at room temperatures. At intervals of
4 h, 0.1 mL of the upper layer of the bacterial suspension was
transferred to another tube with 3.9 mL of PBS, and the
absorbance (A) was measured at 600 nm. Autoaggregation
percentage is expressed as 1 (At/A0) 100, where At
represents the absorbance at different sampling times, and
A0 the absorbance at t = 0.
Motility assays
Swimming and swarming motilities were analysed according
to the methods described by De´ ziel et al. (2001). Ensifer
fredii strain HH103 was grown on soft yeast-mannitol agar.
FEMS Microbiol Lett 259 (2006) 67–73
Data analysis
All the experiments were carried out in duplicate. One-way
ANOVA was used to analyze the results of the inoculum size
assay, using Statistix 7.0. When the analysis of variance
showed significant treatment effect, the least significant
differences test (LSD, P o 0.05) was applied to make comparisons between the means.
Results
Attachment of different rhizobacteria to legume
roots
All the strains used in this work have been tested for their
capacity to attach to both bean and soybean roots, under
control and saline conditions (Tables 1 and 2). When
moderately saline conditions were tested, plantlets and
bacteria were grown in the presence of 50 mM NaCl.
Attachment of the PGPR strains Pseudomonas fluorescens,
A. brasilense, and C. balustinum to both legume roots was,
in general, not affected in the presence of moderate salt
concentration, but C. balustinum showed a better attachment capacity under saline conditions; moreover, P. fluorescens WCS417r and C. balustinum Aur9 cells did show a
significant (P o 0.10) preferential adhesion to soybean
roots.
Adhesion of rhizobial strains to specific (E. fredii strains/
soybean and R. tropici, R. etli/bean) and nonspecific legume
c 2006 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. No claim to original Spanish government works
70
M. Albareda et al.
roots was studied (Tables 2 and 3). Ensifer fredii strains
SMH12 and HH103 showed a low degree of root adhesion,
in comparison with the other rhizobial and PGPR species;
moreover, the attachment to soybean roots grown under
saline conditions significantly (P o 0.10) declined in relation to control roots. Of the rhizobial bean-nodulating
species, R. tropici strain CIAT899 showed a higher degree of
root attachment than did R. etli strain ISP42; however, there
was no differences between species in the attachment to
bean or soybean roots (nonspecific legume) (data not
shown). The presence of salt significantly reduced the
adhesion of R. etli to bean roots (Table 2).
Effect of culture age, inoculum size, and pH on
root attachment
The effect of culture age of the inoculant strains on root
attachment was investigated with specific rhizobia/legume
combinations and with C. balustinum Aur9 on both legume
species (Table 3). In general, bacterial cells at stationary phase
showed a higher capacity to adhere to roots, but in only two
of six cases studied (C. balustinum/Phaseolus and R. tropici/
Phaseolus) was the rate of adhesion significantly higher
(P o 0.10). The effect of the size of the inoculum of R. tropici
CIAT899 and E. fredii SMH12 strains on the attachment to
bean and soybean roots, respectively, was tested (Table 4). In
the case of SMH12, a significantly higher number of rhizobia
attached to roots when the applied inoculum was in the range
of 108–109 CFU mL 1 compared with diluted inocula
(107 CFU mL 1). More-dilute inocula (105–106 CFU mL 1)
gave levels of attachment below quantifiable limits (BQL). In
contrast, saturated cultures of CIAT899 led to significantly
fewer attached cells, with 105–106 CFU mL 1 giving a maximum level of root adhesion.
The effect of pH on the attachment of three rhizobacteria
to the two legumes was studied. Both specific and nonspecific rhizobial strains and C. balustinum were selected for
these studies. The pH of the incubation buffer had a slight
effect on the bacterial attachment to legume roots. At acidic
pH (5.5), there was in general a lower ratio of adhesion to
roots, but with some exceptions (SMH12/Phaseolus and
Aur9/Glycine) (data not shown).
Bacteria traits associated with adhesion
In order to elucidate whether some bacterial traits might be
responsible for the low degree of rhizobial root attachment,
we performed studies of autoaggregation and flagella-associated movement, as bacterial properties linked to surface
adhesion. There was no correlation between root attachment and the autoaggregation ability of the studied bacteria.
The highest percentage of autoaggregation was shown by E.
fredii strains SMH12 (47–62%) and HH103 (65–88%) after
24 h of incubation; on the other hand, these strains had
shown the lowest degree of attachment. For the other
bacterial species, the percentage of autoaggregation varied
from 30% to 40%.
All the studied bacteria showed both modes of flagellaassociated motilities. Swimming motility (owing to the
presence of polar flagella) was determined on plates (0.3%
agar). In the rhizobial strains, the greatest motility was
observed with R. etli strain ISP42 (90 mm) and E. fredii
strain HH103 (80 mm) cells. The rhizobacteria P. fluorescens
showed the greatest movement (75 mm), and C. balustinum
showed a dispersal growth from the point of inoculation
without clear growth rings. Swarming motility (owing to the
presence of lateral flagella) was determined on 0.5% agar
plates. All the strains showed a very uniform swarming
ability, with growth rings ranging from 10 to 30 mm.
Attachment of rhizobial strains to preinoculated
roots with C. balustinum
The effect of the presence of C. balustinum (Aur9) in the
rooting system of bean and soybean plantlets was investigated, before the rhizobial attachment assay using specific
rhizobia/legume combinations, under control and saline
conditions [Tables 5(a) and (b)]. Strain Aur9 was added to
the plant rooting medium 14 h (bean) or 48 h (soybean)
before the attachment assay. The presence of Aur9 (ranging
Table 3. Effect of bacterial growth phase on the attachment of rhizobacterial strains
Bacterial strains
Growth phase
Phaseolus vulgaris
Stationary
Exponential
Glycine max
Stationary
Exponential
Chryseobacterium balustinum
(Aur9)
Rhizobium tropici
(CIAT899)
Rhizobium etli
(ISP42)
Ensifer fredii
(SMH12)
Ensifer fredii
(HH103)
6.23 0.01a
5.31 0.20b
5.93 0.27a
3.82 0.58b
5.59 0.30a
4.96 0.49a
nt
nt
nt
nt
6.37 0.12a
5.11 0.94a
nt
nt
nt
nt
5.86 0.22a
5.21 0.42a
5.47 0.01a
4.71 0.30a
Data represent mean log10 SD values of attached bacteria, from at least two independent assays. Significant differences (P o 0.10) between values
within a column are indicated by different letters.
nt, not tested.
c 2006 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. No claim to original Spanish government works
FEMS Microbiol Lett 259 (2006) 67–73
71
Attachment of rhizospheric bacteria to bean and soybean roots
103–104 CFU mL 1) did not impair the capacity of beannodulating species to attach to bean roots. In the case of
E. fredii strains, the presence of Aur9 (104–106 CFU mL 1)
lessened their capacity of attachment to soybean roots;
however, under saline conditions, the adhesion of the
SMH12 strain to preinduced soybean roots was significantly
better than to control roots. The adhesion of the CIAT899
strain to preinduced bean roots, under saline conditions,
was also better. In the case of the R. etli ISP42 strain, there
was no variation in its attachment capacity under the
different assay conditions.
Discussion
PGPRs have drawn much attention in recent years because
of their contribution to the biological control of plant
pathogens and the improvement of plant growth. Inoculation of plants with ‘dual’ microbial inoculants, or even a
consortium of them, is becoming more important in a
framework of sustainable agriculture for the advantage their
beneficial effects afford, providing there is no competition
Table 4. Effect of bacterial inoculum size on the attachment of rhizobial
strains to specific legume–host roots
Inoculum (Lg CFU mL 1)
Strains
9
8
7
6
5
SMH12
CIAT899
52.4b
88.8c
84.3a
260.8b
11.1c
245.8b
BQL
925.5a
BQL
803.8a
Data are means of two replicates. Significant differences (P o 0.05)
between values within each row are indicated by different letters.
BQL, below quantifiable limits.
between inoculants. However, the practical use of these
beneficial bacteria sometimes fails because of their inability
to colonize the rhizosphere or the rhizoplane of inoculated
plants. Colonization of roots by inoculant strains thus
appears to be a critical step in the interaction between
beneficial bacteria and host plants. Attachment of rhizobia
to host roots seems to be the first requisite step in infection
and nodulation (Bohlool & Schmidt, 1974), and is considered one of the physiologically important characteristics in
determining competitive ability among rhizobial strains
(Smith & Wollum, 1991).
Root colonization is defined as a complex phenomenon
depending upon several biotic and abiotic factors. In this
work, we have defined a hydroponic system to grow plants
under bacteriologically controlled conditions, allowing
changes in the rooting media (saline or control conditions),
the controlled addition of bacterial inoculants, or pH
changes. The attachment assay used in this work is similar
to that of Dardanelli et al. (2003), and the values for
bacterial attachment presented here can be considered those
of tightly attached bacteria. Chabot et al. (1996) have
described rhizobial strains that were better colonizers of
maize and lettuce roots than were other PGPR bacteria; our
results, however, indicated that the studied PGPRs are in
general better colonizers of the two legumes than are the
specific rhizobial species, except Rhizobium tropici/bean
roots. An attempt was made to correlate the low degree of
rhizobial attachment with physicochemical characteristics of
the cell surface by measuring the autoaggregation of the
studied rhizobacteria, as well as the flagella-associated
motilities. There is a relationship between the bacterial
ability for autoaggregation and that of adhesion to different
Table 5. Attachment of (a) bean-nodulating and (b) soybean-nodulating strains to non- or preinoculated roots with Chryseobacterium balustinum
strain Aur9
Rhizobial strains
Presence of C. balustinum
(a) Bean-nodulating strains
Non
1 Aur9
Plant growth conditions
Rhizobium tropici CIAT899
Rhizobium etli ISP42
Control
Salt
Control
Salt
6.34 0.08ab
5.92 0.27b
6.33 0.10ab
6.56 0.01a
5.42 0.11a
4.88 0.21a
4.78 0.34a
5.40 0.42a
Rhizobial strains
Presence of C. balustinum
(b) Soybean-nodulating strains
Non
1Aur9
Plant growth conditions
Ensifer fredii HH103
Ensifer fredii SMH12
Control
Salt
Control
Salt
5.13 0.34a
3.45 0.06b
4.23 0.26b
nt
5.24 0.08a
3.64 0.16d
5.03 0.02b
4.18 0.02c
Data represent mean log10 SD values of attached bacteria, from at least two independent assays. Significant differences (P o 0.10) between values
within a column are indicated by different letters.
nt, not tested.
FEMS Microbiol Lett 259 (2006) 67–73
c 2006 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. No claim to original Spanish government works
72
surfaces (Zaady & Okon, 1990; Kos et al., 2003). Our data
agree with those of Zaady & Okon, as they have shown that
Azospirillum cell treatments leading to strong inhibition of
aggregation culminate in the greatest adsorption to maize
roots. In our case, the highest autoaggregation percentage
was shown by E. fredii strains, which showed the lowest
degree of root attachment. However, our results do not agree
with those obtained by Kos et al. (2003) using Lactobacillus
strains. It has been suggested that anionic repulsions
between root and rhizobia may explain why only a small
proportion of a rhizobial population attaches to legume
roots (Vesper & Bauer, 1985; Caetano-Anolles & Favelukes,
1986). In addition, bacterial cell movements because of
flagella have been linked to adherence of some Gramnegative gastrointestinal bacteria to epithelial cells in vitro
and to other inert surfaces. We have determined the modes
of flagella-associated motilities, owing to polar and lateral
flagella, and found no defective swimming or swarming
motilities that might explain the differences in root attachment capacity of the studied bacteria.
The level of rhizobial adhesion to the specific or nonspecific host legume roots was similar for all studied species,
although bean-nodulating rhizobia are better colonizers
than soybean-E. fredii microsymbionts. Nevertheless, in our
study the proportion of attached cells with respect to
inoculated levels is much lower than that reported in other
studies (Vesper & Bauer, 1985, 1986). These results reinforce
the concept of lack of specificity of host legume exudates for
their homologous rhizobial microsymbionts, as has been
largely demonstrated (Pueppke, 1984; Vesper & Bauer, 1985;
Dolhem-Biremon et al., 1993). A nonspecific mechanism,
independent of the symbiotic properties, must contribute to
root attachment of rhizobia, as the same colonization level
has been reported for P. fluorescens and Sinorhizobium
meliloti in alfalfa roots (Villacieros et al., 2003). However, a
certain specificity of plant exudates to their bacterial isolates
have also been proposed (Mandimba et al., 1986; BacilioJime´ nez et al., 2003).
It has been reported that culture age affects the extent of
bradyrhizobial attachment to soybean roots, and that it is
strain-dependent (Smith & Wollum, 1991). We have also
found that culture age has a distinct effect on root attachment of certain strains, and there was a general and positive
tendency for a greater attachment level in stationary cells.
Although bacterial cultures were twice washed and centrifuged with buffer, so that extracellular polymeric substances
should have been removed, when cells enter the stationary
phase of growth, other morphological and biochemical
changes occur that might modify the degree of attachment.
Moreover, acidification (pH 5.5) of the attachment buffer
did not greatly affect the adhesion of studied bacteria in
comparison with control buffer (pH 7.5). Possible modifications of the bacterial cell surface during 1 h of incubation
2006 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. No claim to original Spanish government works
c
M. Albareda et al.
might account for the slight differences observed, as changes
in root surface are probably negligible; in fact, the final pH
of the rooting medium usually decreases after 1 week of root
development. Using two organic buffering systems, Smith &
Wollum (1993) found an optimum pH range (6.0–6.5) for
the attachment of Bradyrhizobium japonicum USDA110 to
soybean roots. By contrast, root surface modifications
occurring when 50 mM NaCl was added to the rooting
system might be strong grounds for some differences in
bacterial root attachment under saline conditions.
Root attachment was dose-dependent in the case of E.
fredii SMH12 strain, and optimum adhesion was found at
a concentration of 108–109 CFU mL 1, in agreement with
other results (Jjemba & Alexander, 1999). However, in the
case of R. tropici CIAT899, a large inoculum gave poor
attachment to bean roots. Differences in the optimum
attachment of these strains with varying inoculum size
might be attributable to differential bacterial traits rather
than to a limitation of binding sites in these root-segment
assays, as most of the attachment trials have been performed
with saturated bacterial inocula or in the presence of other
bacteria (preinoculated C. balustinum roots), and small
percentages of rhizobial populations were consistently
found to adhere firmly to roots.
The presence of C. balustinum Aur9 strain in the rooting
media 15–20 h before rhizobial-attachment assays did not
affect the adhesion of bean-nodulating species, but did
lessen the attachment of E. fredii strains; perhaps the higher
level of Aur9 in the soybean rooting media was the reason
for this difference.
The study has demonstrated that the coinoculation of
Aur9 and rhizobial strains provides the same growth capacity in root exudates as that of single inocula, but further
studies of proper root colonization dynamics, using whole
root systems, are necessary to reveal the actual binding
points on the roots, and to demonstrate persistence of the
studied strains in the rhizosphere.
Acknowledgements
´ y
This research was founded by the Ministerio de Educacion
Ciencia (Spain). Project: AGL2002-04188-CO6.
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