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Mycorrhiza (2003) 13:249–256
DOI 10.1007/s00572-003-0223-z
ORIGINAL PAPER
Astrid Vivas · Adriana Marulanda ·
Juan Manuel Ruiz-Lozano · Jos Miguel Barea ·
Rosario Azcn
Influence of a
Bacillus
sp. on physiological activities
of two arbuscular mycorrhizal fungi and on plant responses
to PEG-induced drought stress
Received: 24 May 2002 / Accepted: 8 January 2003 / Published online: 15 February 2003
Springer-Verlag 2003
Abstract The effects of bacterial inoculation (Bacillus
sp.) on the development and physiology of the symbiosis
between lettuce and the arbuscular mycorrhizal (AM)
fungi Glomus mosseae (Nicol. and Gerd.) Gerd. and
Trappe and Glomus intraradices (Schenck and Smith)
were investigated. Plant growth, mineral nutrition and
gas–exchange values in response to bacterial inoculation
after PEG–induced drought stress were also evaluated. In
AM plants, inoculation with Bacillus sp. enhanced fungal
development and metabolism, measured as succinate
dehydrogenase (SDH) and alkaline phosphatase (ALP)
activities, more than plant growth. Under non-stressed
conditions, G. intraradices colonization increased all
plant physiological values to a higher extent when in dual
inoculation with the bacterium. Under stress conditions,
the bacterium had an important stimulatory effect on G.
intraradices development. Under such conditions, the
effects of the bacterium on photosynthetic rate, water use
efficiency (WUE) and stomatal conductance of lettuce
plants differed with the fungus species. Plant-gas ex-
change was enhanced in G. intraradices- and reduced in
G. mosseae-colonized plants when co-inoculated with
Bacillus sp. Thus, the effects of each fungus on plant
physiology were modulated by the bacterium. Stress was
detrimental, particularly in G. intraradices-colonized
plants without the bacterium, reducing intra and extrarad-
ical mycelium growth and vitality (SDH), as well as
plant-gas exchange. Nevertheless, Bacillus sp. inoculation
improved all these plant and fungal parameters to the
same level as in non-stressed plants. The highest amount
of alive and active AM mycelium for both fungi was
obtained after co-inoculation with Bacillus sp. These
results suggest that selected free-living bacteria and AM
fungi should be co-inoculated to optimize the formation
and functioning of the AM symbiosis in both normal and
adverse environments.
Keywords Alkaline phosphatase · AM fungus ·
Bacterium co-inoculation · PEG-induced drought stress ·
Succinate dehydrogenase
Introduction
The rhizosphere is a site at which symbiotic and non-
symbiotic microorganisms can interact (Jeffries and Barea
1994). Ubiquitous arbuscular mycorrhizal (AM) fungi and
plant-growth-promoting rhizosphere bacteria are compo-
nents of natural systems that benefit the host plant (Barea
and Jeffries 1995). As a result of microbial-plant associ-
ations, crop production can be increased and mixed
inocula have been recommended for re-vegetation pur-
poses (Bowen and Rovira 1999). The cooperation of AM
fungi and bacteria in nutrient uptake by plants may be due
to specific attributes of microorganisms, such as the
ability of certain bacteria to stimulate mycorrhizal
formation and development (Azcn-Aguilar and Barea
1985; Garbaye 1994), as well as mycorrhizal effects on
the associated bacterial population (Amora-Lazcano and
Azcn 1997; Amora-Lazcano et al. 1998). There is a
growing interest in improving understanding of the
diversity and significance of microbial populations in
soil and their involvement in nutrient cycling. The
manipulation of certain bacteria and AM fungi is
important with regard to sustainability issues (Bowen
and Rovira 1999; Barea et al. 2002).
Many authors have correlated the plant growth
responses to mycorrhization with high amounts of AM
roots and fungal physiological activity, which is closely
related to mycorrhizal functioning (Tisserant et al. 1993;
A. Vivas · A. Marulanda · J. M. Ruiz-Lozano · J. M. Barea ·
R. Azcn ())
Departamento de Microbiologa del Suelo y Sistemas Simbiticos,
Estacin Experimental del Zaidn, CSIC,
Profesor Albareda 1, 18008 Granada, Spain
e-mail: Rosario.Azcon@eez.csic.es
Tel.: +34-958-121011
Fax: +34-958-129600
Guillemin et al. 1995). It is well known that microbial
activities can affect the formation of mycorrhiza (Azcn
et al. 1978; Azcn 1989), but there is no information
about the effect of bacterial inoculation on metabolic
characteristics of AM fungi.
AM colonization is normally quantified by assessing
the level (frequency and intensity) of fungal infection in
plant roots after trypan blue (TB) staining (Phillips and
Hayman 1970). However, Vierheilig and Ocampo (1989)
reported that TB staining was not a good indicator of AM
efficiency and, in AM studies related to the functional
aspect of symbiosis, vital staining techniques have been
suggested (Tisserant et al. 1993; Boddington and Dodd
1998). Succinate dehydrogenase (SDH) and alkaline
phosphatase (ALP) activities, linked to active metabolic
fungal performance, have been proposed as useful indices
for the analysis of efficient arbuscular endomycorrhizal
infection (Smith and Gianinazzi-Pearson 1990; Tisserant
et al. 1993; Vzquez et al. 2000).
Mycorrhizal symbiosis is considered a key factor in
helping plants cope with adverse environmental condi-
tions (Jeffries and Barea 1994). Although an effect of AM
endophytes on drought alleviation has been reported
(Ruiz-Lozano et al. 1995a, 1995b, 2001), no information
is available on the ability of plants dually inoculated with
AM fungi and mycorrhiza-helper bacteria to support
drought stress. The bacterium assayed here (Bacillus sp.)
was isolated from a desert area (Alicante, Spain) and
shown to stimulate growth of red clover plants (Vivas et
al., unpublished results). An experimental area was
chosen within a desertification-threatened region in
southeast Spain for studying the role of the most common
natural saprophyte and symbiotic microbial isolates in
sustaining vegetation cover in such stressed environ-
ments. The AM fungi selected for this study were Glomus
mosseae and G. intraradices, which are well adapted to
the dry conditions of Mediterranean areas (Ruiz-Lozano
et al. 1995a, 1995b).
The aim of this study was to determine the effect of
inoculation with a Bacillus sp. from a desert area on the
growth, vitality and activity of the above-mentioned
fungi. An additional objective was to determine the
possible importance of the bacterium in combined
bacterial and mycorrhizal inocula for plant tolerance to
drought stress and to define the possible mechanisms
involved.
Materials and methods
Experimental design and statistical analysis
At sowing time, plants were inoculated with either G. mosseae or G.
intraradices and/or Bacillus sp. The bacterium and each of the
mycorrhizal fungi were assayed alone or in dual fungus-bacterium
combinations. Thus, there were five microbial treatments plus one
control treatment without inoculation. Ten replicates of each
treatment were made giving a total of 60 pots. Half of the pots
were cultivated without PEG application and half exposed to PEG
prior to harvesting.
Data were subjected to an analysis of variance (ANOVA)
followed by Duncan’s multiple range test (Duncan 1955). Percent-
age values were arcsin transformed before statistical analysis.
Soil and biological material
Lettuce (Lactuca sativa L. cv Romana) was grown for 2 months in
300 g sand/vermiculite/sepiolite (1:1:1) inert medium, previously
washed and sterilized by autoclaving.
The bacterial inoculum was isolated from a desert soil in
Alicante province (Spain) using standard techniques following
serial dilutions of the soil. The inoculum was grown on a rotary
shaker (150 rpm) at 28C for 28 h in a 250-ml flask containing
50 ml of nutrient broth (8 g l–1) solution. Aliquots (1 ml) of the
bacterial culture containing 108CFU ml–1 were added to each pot.
Mycorrhizal fungal inoculum from each endophyte was multi-
plied in an open-pot culture of Lactuca sativa L. and consisted of
soil, spores, hyphae and AM root fragments. The AM species were
G. mosseae (Nicol. and Gerd.) Gerd. and Trappe, isolate BEG 122
and G. intraradices (Schenck and Smith) isolate BEG 121. Aliquots
(5 g) of each inoculum, with similar infective characteristics (an
average of 50 propagules g-1 according to the most probable
number test), were placed below the seeds of Lactuca sativa L. This
amount of inoculum was selected in preliminary tests as producing
an optimal infection level according to the total amount of soil in
the pot. Non-mycorrhizal treatments received the same quantity of
autoclaved inoculum together with a 2-ml aliquot of a filtrate (less
than 20 m) of the AM inoculum, in an attempt to provide a general
microbial population free of AM propagules. Two seeds were sown
and thinned after emergence to one seedling per pot.
Growth conditions
The plants were grown in a controlled-environment chamber under
conditions of 50% relative humidity, day and night temperatures of
27C and 18C, respectively, and a photoperiod of 14 h. Photo-
synthetic photon flux density (PPFD) was 500 mol m–2 s–1 as
measured with a light meter (LICOR, model LI-188B).
Drought stress was induced by adding PEG solution (15% PEG)
to half of the pots at two growing stages (1 week and 24 h before
harvesting) as described elsewhere (Ruiz-Lozano and Azcn 1997),
so that the two PEG applications had accumulative effects. A PEG
solution (purified PEG 6,000 Merck, molecular weight 5,000–
7,000) was used as osmoticum to induce stress. The rest of the pots
were kept as controls without PEG.
The plants were given a nutrient solution (Hewitt 1952) at half-
P concentration with the pH adjusted to 6.8–7. This mineral
solution has been found to give high levels of infection in plants
inoculated with G. mosseae and G. intraradices (Ruiz-Lozano and
Azcn 1996). Mineral solution was supplied twice a week (25 ml
per pot) to maintain the required water and nutrient levels
throughout the experiment. A rock phosphate source from Morocco
with 19 mg P2O5kg–1 (Olsen and Dean 1965) was applied to each
pot by mixing 1 g with the sand/vermiculite/sepiolite medium
before sterilizing. This soil-less substrate was selected as growth
medium since it provides little benefit to the AM host plant and
thus allows a more direct interpretation of the mechanisms of the
bacterial-fungal interaction.
Parameters measured
Before harvest, the CO2exchange rate, transpiration rate, sub-
stomatal cavity CO2concentration and instantaneous water use
efficiency (WUE) were measured on the fourth leaf below the apex
of each plant. Atmospheric CO2was measured 5 m above ground
level. PPFD was 1,180 mmol m–2 s–1, which ensured that no
limitation in photon irradiance occurred (Long and Hallgren 1987).
Light was provided by a halogen lamp (General Electric 300 PAR
56/WFL). A model LCA-3 portable integrated infrared CO2
250
analyzer (Analytical Development Co., Hoddesdon, UK) was used
for these determinations. Measurements were made 2 h after the
light was turned on.
At harvest (8 weeks after planting), the root system was
separated from the shoot and the fresh and dry weights of each were
recorded. The roots were carefully washed and then divided into
three batches: one was stained by the normal non-vital TB staining
of all fungal tissues (Phillips and Hayman 1970) and the others
were used for histochemical vital staining of the mycorrhizal roots
to measure living (SDH) and functional (ALP) mycorrhizal fungal
development.
SDH activity was revealed by the procedure described by Smith
and Gianinazzi-Pearson (1990). The roots were immersed in a
freshly made solution of 0.2 M Tris-HCl pH 7.0, 2.5 M sodium
succinate 6-hydrate, 4 mg ml-1 nitro blue tetrazolium, 5 mM MgCl2.
Root fragments were stained overnight at room temperature and
then rinsed for 15–20 min in a 3% active chlorine solution of
sodium hypochlorite.
ALP was determined according to the procedure described by
Tisserant et al. (1993). The roots were immersed in a freshly made
solution containing 50 mM Tris-citric acid, pH 9.2, 1 mg ml-1 a-
naphthyl acid phosphate (monosodium salt), 0.05% MgCl2anhy-
dro, 0.05% MnCl2tetrahydrate and 1 mg ml-1 fast blue RR salt.
Root fragments were stained overnight at room temperature and
then rinsed for 15–20 min in a 1% active chlorine solution of
sodium hypochlorite.
Mycorrhizal development, after either non-vital or vital staining
procedures, was evaluated by the method of Trouvelot et al. (1986)
(for more information, see http://www.dijon.inra.fr/bbceipm/Mych-
intec/Mycocalc-prg/). An estimate of the length of root colonized by
the fungus (the colonization frequency, F%) is given as the ratio
between colonized root fragments and the total number of root
fragments observed. The colonization intensity (m%) is an estimate
of the amount of root cortex that became mycorrhizal, relative only
to the mycorrhizal root fraction, while M% is the colonization
intensity relative to the whole root system. Arbuscule abundance (a%
and A%) is an estimate of arbuscule richness in the mycorrhizal root
fraction and in the whole root system, respectively.
The extraradical mycelium in the soil was determined by an
adaptation of the method described by Jones and Mollison (1948).
Briefly, 1 g of dry soil was treated with sodium hexametaphosphate
and trypan blue (0.05%) in lactic acid. The sample was heated in a
water bath at 90C for 30 min and then sieved through a 50-m-
mesh sieve. The remaining mycelium was mixed with bacterio-
logical agar for quantification using a gridline intersection method
described by Newman (1966).
Proline content in leaves and roots was determined by
colorimetry (Bates et al. 1973). Concentrations of N (micro-
Kjeldahl), P (Olsen and Dean 1965) and K (Lachica et al. 1973) in
the shoots were measured.
Molecular identification of the bacterial strain
Total DNA from the bacterial isolate was obtained as described by
Giovannetti et al. (1990) and characterized by 16S ribosomal DNA
sequence analysis. PCR was carried out with the eubacterial
primers 27f and 1495r (Lane 1991), located at the 5’ and 3’ ends of
the ribosomal rDNA sequence, respectively, which enabled us to
amplify almost the entire gene. Amplification reactions were done
in a 20-l volume containing 0.5 M of each primer, 100 M
dNTPs, 1 PCR buffer (Sigma, St. Louis, Miss., USA), 2.5 mM
MgCl2, 10 ng of genomic DNA and 0.25 U Taq DNA polymerase
(Sigma). A Perking-Elmer/Cetus DNA Thermal Cycler was used
with the following parameters: initial denaturation at 95C for
4 min, followed by 30 cycles of denaturation at 94C for 30 s,
annealing at 56C for 45 s, elongation at 72C for 1 min and a final
elongation at 72C for 5 min. The amplified DNA was purified
following electrophoresis through a 1.2% agarose gel with the
QIAEX II Gel Extraction Kit (Qiagen, Hilden, Germany) and
cloned into the pGME plasmid (Promega) for sequencing. Database
searches for 16S rDNA sequence similarity, using FASTA and
BLAST algorithms, unambiguously identified the bacterial isolate
as a member of the genus Bacillus. However, the exact species must
still be elucidated by complementary studies.
Results
The inert mixture used in this study as rooting medium was
a good substrate for mycorrhizal performance. Mycorrhizal
plants grew better than non-mycorrhizal plants and devel-
oped abundant AM (Fig. 1, Table 1). Inoculation with
Bacillus sp. alone also increased shoot and root growth and
proved to be the most effective treatment for enhancing
root growth in stressed plants (Fig. 1). Plants colonized by
G. intraradices alone showed the highest shoot growth,
particularly under non-stressed conditions (Fig. 1). In PEG
treatments, the two AMF increased shoot biomass with
similar efficiencies when inoculated independently. Bacil-
lus sp. increased plant growth more effectively in non-
mycorrhizal plants (Fig. 1).
In the absence of bacteria and PEG, the total infective
parameters (TB staining) were highest in plants colonized
by G. intraradices (Table 1). Nevertheless, the same
infective values were attained when Bacillus sp. and G.
mosseae were co-inoculated. Greatest infectivity was not
Fig. 1 Shoot and root fresh weight (g) in lettuce plants subjected or
not to PEG-induced drought stress. Treatments are described as C
(control), B(Bacillus sp.), Gm (G. mosseae), Gi (G. intraradices),
Gm+B (G. mosseae +Bacillus sp.) and Gi+B (G. intraradices +
Bacillus sp.). Means not followed by a common letter differ
significantly (P=0.05) from each other according to Duncan’s
multiple range test (n=5)
251
always related to mycorrhizal response (Table 1, Fig. 1).
Bacterial inoculation did not increase shoot growth in
mycorrhizal plants but did enhance mycorrhizal infective
parameters compared with those of roots colonized by G.
mosseae or G. intraradices alone (Table 1). The bacteria
enhanced both live (SDH) and active (ALP) mycorrhizal
fungal biomass (Table 1) in all treatments. Bacterial
inoculation almost doubled ALP activity in both types of
mycorrhizal root. Highest ALP activity was found in the
absence of PEG in plants co-inoculated with Bacillus sp.
and G. intraradices.
With regard to AM-colonizing parameters, G. in-
traradices-colonized roots showed higher ALP activity
than G. mosseae-colonized roots under stress conditions,
despite the fact that vital SDH activity was higher in G.
mosseae- than in G. intraradices-colonized roots. Thus,
the ratio of activity (ALP) to vitality (SDH) in G.
intraradices-colonized plants was higher than in G.
mosseae-colonized plants. Bacterial inoculation enhanced
the metabolic characteristics of G. intraradices under
stress conditions to a greater extent than in the absence of
PEG. Most noticeable was that Bacillus sp. widely
enhanced AM colonization and fungal metabolic activity
in the mycorrhizal tissue (Table 1). Lettuce roots
colonized by either of the AMF reached maximum
infection and live and active mycelium in the presence
of Bacillus sp.
Concerning plant-gas exchange, the transpiration rate
was hardly affected by the microbial treatment, both
under stress and non-stress conditions (Table 2). In
contrast, the photosynthetic rate, WUE and stomatal
conductance were considerably increased by both myc-
orrhizal fungi under non-stress conditions. The bacterial
treatment had different effects on these parameters
depending upon the associated Glomus species. Co-
inoculation with Bacillus sp. increased photosynthesis,
WUE and stomatal conductance in G. intraradices-
colonized plants but not in G. mosseae-colonized ones.
However, under stress conditions, G. mosseae-colonized
plants showed the highest gas-exchange values (Table 2).
Drought stress decreased plant-gas exchange to a
higher extent in plants colonized by G. intraradices than
in those colonized by G. mosseae. Under such conditions,
considerable differences were found in CO2exchange and
WUE between the least effective (G. intraradices) and the
most effective (G. mosseae) fungal species (Table 2). Co-
inoculation with Bacillus sp. increased photosynthesis,
WUE and stomatal conductance in G. intraradices-
colonized plants relative to mycorrhizal inoculation alone,
but not in the case of G. mosseae-colonized plants.
Under non-stress conditions, the proline content in
leaves reached its maximum value in plants dually
inoculated with G. intraradices and Bacillus sp. Under
stress conditions, maximum proline accumulation was
reached in the leaves of plants inoculated with G. mosseae
alone (Fig. 2).
The highest amount of extraradical mycelium pro-
duced by G. intraradices in the absence of PEG correlated
with the highest ability of this fungus for intraradical
colonization. Bacillus sp. did not affect extraradical
mycelium development. In contrast, under stress condi-
tions, the bacterium increased the length of the mycelium
produced by G. intraradices (Fig. 3).
The N, P and K contents were also affected by the
microbial treatments (Fig. 4). Under non-stress condi-
tions, N was only significantly increased by dual G.
mosseae-Bacillus sp. inoculation, while no effect was
observed under stress conditions. Plant P content was
Table 1 Effect of Bacillus sp. on arbuscular mycorrhizal coloni-
zation as measured by trypan blue (TB), succinate dehydrogenase
(SDH) or alkaline phosphatase (ALP) staining in lettuce roots
subjected or not to PEG-induced drought stress. Treatments were
Gm (G. mosseae), Gi (G. intraradices), Gm+B (G. mosseae +
Bacillus sp.) and Gi+B (G. intraradices +Bacillus sp.).Within each
parameter, means with the same letter are not significantly different
according to Duncan’s multiple range test (P=0.05, n=3) (%F the
colonization frequency, given as the ratio between colonized root
fragments and the total number of root fragments observed, %m
colonization intensity, as an estimate of the amount of root cortex
that became mycorrhizal, relative only to the mycorrhizal root
fraction, %M the colonization intensity relative to the whole root
system, %a arbuscule abundance as an estimate of arbuscule
richness in the mycorrhizal root fraction, %A arbuscule abundance
as an estimate of arbuscule richness in the whole root system)
Staining %F %M %m %a %A
TB
No PEG
Gm 98 a 46.4 c 47.3 c 68.0 c 31.5 c
Gm+B 97 a 87.6 a 90.3 a 98.0 a 85.8 a
Gi 99 a 73.4 ab 74.1 ab 84.0 b 61.6 b
Gi+B 99 a 87.6 a 88.5 a 99.0 a 86.7 a
PEG
Gm 97 a 44.1 c 45.5 c 88.0 ab 38.8 bc
Gm+B 97 a 69.9 b 72.0 b 89.8 ab 62.8 b
Gi 99 a 42.4 c 42.8 c 84.0 b 35.6 c
Gi+B 97 a 72.0 ab 74.2 ab 100.0 a 72.0 a
SDH
No PEG
Gm 99 a 33.0 c 33.3 c 67.4 c 22.2 d
Gm+B 99 a 73.0 a 73.7 a 97.1 a 70.9 a
Gi 98 a 69.7 a 71.1 a 80.5 b 56.1 b
Gi+B 97 a 76.7 a 79.0 a 97.7 a 75.0 a
PEG
Gm 97 a 42.9 c 44.2 c 87.7 b 37.4 d
Gm+B 98 a 60.4 b 61.6 b 83.1 b 50.2 b
Gi 98 a 34.7 c 35.4 c 83.8 b 29.1 cd
Gi+B 99 a 71.7 a 72.4 a 96.1 a 68.9 a
ALP
No PEG
Gm 97 a 18.3 c 18.8 c 61.4 c 11.2 d
Gm+B 97 a 34.3 b 35.3 b 71.4 b 24.5 b
Gi 99 a 18.5 c 18.7 c 84.8 a 15.7 c
Gi+B 97 a 48.2 a 49.6 a 82.2 a 39.7 a
PEG
Gm 99 a 17.5 c 17.6 c 60.8 c 10.6 d
Gm+B 98 a 47.4 a 48.3 a 76.7 b 36.4 a
Gi 98 a 30.0 b 30.6 b 62.1 c 18.5 c
Gi+B 98 a 42.2 a 43.0 a 80.9 ab 34.1 a
252
increased by all the microbial treatments compared with
the uninoculated controls under non-stress conditions.
Again, no effect on P was found under stress conditions.
The K content under non-stress conditions was also
increased by all treatments. In contrast, under stress
conditions, plants inoculated with G. intraradices alone or
those dually inoculated with G. mosseae and Bacillus sp.
had the highest K content.
Discussion
We have shown that a rhizospheric bacterium isolated
from a desert soil and identified as Bacillus sp. can
influence the development and activity of two Glomus
species. No information is available about the mode of
action of mixed inocula on plant and/or fungal physio-
logical status or effects on plant performance under
drought stress. Under our experimental conditions, the
bacterium was as effective as G. mosseae in increasing
shoot growth and nutritional status. It also stimulated
Fig. 2 Proline content (mg g
FW–1) in lettuce plants subject-
ed or not to PEG-induced
drought stress. Treatments as in
Fig. 1. Means not followed by a
common letter differ signifi-
cantly (P=0.05) from each other
according to Duncan’s multiple
range test (n=5)
Table 2 Effect of Bacillus sp. and PEG-induced drought stress on
transpiration rate (mmol H20m
1 s1), photosynthetic rate (mmol
CO2m2 s1), water use efficiency (mmol CO2mol H201) and
stomatal conductance (mol H2Om
1 s1) of lettuce plants.
Treatments as in Table 1 plus C(control) and B(Bacillus sp.).
Within each parameter, means with the same letter are not
significantly different according to Duncan’s multiple range test
(P= 0.05, n=5)
Microbial treatments Transpiration Photosynthetic rate Water use efficiency Stomatal conductance
No PEG
C 2.59 a 8.92 e 3.45 d 7.43 d
B 2.36 b 5.79 f 2.46 e 12.14 c
Gm 2.32 b 10.48 c 4.52 c 14.26 ab
Gi 2.34 b 12.35 b 5.28 b 13.45 b
Gm+B 2.32 b 9.81 d 4.22 c 14.09 ab
Gi+B 2.25 b 12.86 a 5.75 a 15.19 a
PEG
C 2.22 b 4.52 bc 1.79 c 7.95 d
B 2.31 a 3.59 d 1.56 d 12.33 c
Gm 2.06 e 5.50 a 2.67 a 16.15 a
Gi 2.23 b 2.52 e 1.23 e 14.22 b
Gm+B 2.17 cd 4.22 c 1.95 c 15.04 ab
Gi+B 2.12 de 4.73 b 2.23 b 15.95 a
Fig. 3 Extraradical mycelium
production (cm g soil–1)by
mycorrhizal plants subjected or
not to PEG-induced drought
stress. Treatments as in Fig. 1.
Means not followed by a com-
mon letter differ significantly
(P=0.05) from each other ac-
cording to Duncan’s multiple
range test (n=5)
253
considerably the metabolic activity of the intraradical
mycelium developed in G. mosseae- and G. intraradices-
colonized roots, as well as the development of the
extraradical mycelium of G. intraradices. In fact, the
inoculation of AM plants with Bacillus sp. enhanced
fungal development and metabolism (in terms of SDH
and ALP) more than plant growth. The ability of Bacillus
sp. to increase the proportions of both alive and active
intraradical mycelium suggests a direct bacterial effect on
the metabolic status of these fungi. Thus, the production
of active metabolites such as vitamins, amino acids and
growth substances (e.g. indoleacetic acid) by this bacte-
rium (Vivas et al., unpublished results) could directly
stimulate the growth and metabolic capacity of AM
endophytes. The bacterium appears to act as a mycorrhi-
za-helper microorganism (Garbaye 1994; Barea 1997).
Another possibility is that the enhanced photosynthetic
rate found in plants dually inoculated with G. intraradices
and the bacterium affects the translocation of soluble
sugars to host roots, thus increasing fungal growth and
activity in the root (Amijee et al. 1989; Hetrick 1989).
Nevertheless, co-inoculation of this bacterium with G.
mosseae did not increase plant gas-exchange parameters
and, curiously, it enhanced intraradical fungal growth and
metabolism. The specific interactions between the bacte-
rium and each Glomus sp. on plant physiology and
metabolism were independent of drought stress and, thus,
seem not to be related to fungal growth.
The increases in photosynthetic rate, WUE and
stomatal conductance induced by bacterial inoculation
of G. intraradices-colonized plants, and the contrasting
effects on G. mosseae-infected plants, cannot be attribut-
ed to general stimulation by this bacterium of mycelial
growth and metabolic activity in the intraradical fungal
biomass developed by each endophyte. The effect seems
to be due to a specific microbe-microbe interaction that
modulates the effectivity of each AMF on plant physiol-
ogy. The specific microbial compatibilities determined
Fig. 4 N, P and K shoot contents (mg plant–1) in lettuce plant
subjected or not to PEG-induced drought stress. Treatments as in
Fig. 1. Means not followed by a common letter differ significantly
(P=0.05) from each other according to Duncan’s multiple range test
(n=5)
254
were not translated into a higher shoot biomass under the
experimental conditions used. We only found an in-
creased plant capacity for nutrient uptake, perhaps
because of higher fungal metabolic activity.
The lack of response by plants co-inoculated with G.
mosseae and Bacillus sp. may be explained by the fact
that the AM fungus plus the bacterium used plant
carbohydrates during early plant development and, thus,
created a carbon drain on the plant, as reported by
Johnson et al. (1997). In plants colonized with specific
microbial groups, the microbial carbon requirements and
below-ground respiration seem to be higher (Pang and
Paul 1980) and represent an important carbohydrate cost
for the plant. However, in treatments without PEG, the
mycorrhizal association stimulated photosynthetic rate
(see Table 2) and compensated the C cost, as pointed out
by Tinker et al. (1994). Thus, the assimilation, translo-
cation and utilization of fixed C are integrated processes
with a more complex interpretation in mycorrhizal plants
(Smith and Gianinazzi-Pearson 1990).
The decrease in photosynthetic activity as a conse-
quence of drought stress was greater in mycorrhizal plants,
particularly in those colonized by G. intraradices. This
photosynthetic depression caused by stress negatively
affected both extraradical fungal development and in-
traradical activity (SDH). Wright et al. (1998) estimated
that 4–20% of the total photosynthesized C was used by the
AM endophyte, and the fungal consumption of carbohy-
drates can be critical for plant physiological processes
when environmental conditions are limiting. The present
results suggest that, under stress conditions, either the
fungal respiration rate increased in G. intraradices-colo-
nized roots or the high C requirements in these roots
(Olsson and Johansen 2000) decreased more than the
amount of C fixed in tissues colonized by this fungus
(Smith and Smith 1996). Here, the differences in intrarad-
ical colonization levels do not explain differences in the
photosynthetic rate between the mycorrhizal treatments
under stress and non-stress conditions. However, plants
would respond differently to colonization by AM fungi
having different carbon requirements (Dodd et al. 2000).
The difference in the interactions between the bacte-
rium and each AM fungus was also evident in proline
accumulation in plant leaves. Proline accumulation is
involved in osmotic cellular adaptation, but no clear
relationship between proline, AM colonization and envi-
ronmental stress has been found (Ruiz-Lozano et al.
1995a). In this study, the increasing proline content under
PEG-induced drought stress is an indication of adjustment
of leaf osmotic potential, required for enhanced intracel-
lular osmotic balance. Plants exposed to drought stress
usually reduce the stomatal aperture to avoid water loss,
but we found no significant effect of PEG on this
parameter. In contrast, stressed plants increased drought
resistance by maintaining high levels of proline, photo-
synthetic activity and WUE. All these processes are
mechanisms by which plants can cope with drought stress
(Gale and Zeroni 1985; Ruiz-Lozano et al. 1995a).
The relative effectiveness of the two AM fungi results
from the development of arbuscules for nutrient transfer
between the symbionts (Varma and Hoock 1998).
According to our study, both the quantity and activity
of these arbuscules were increased by bacterial inocula-
tion. As a result, changes in nutrient transfer to the plant
and the equilibrium in the transfer of carbohydrates to the
fungus must occur in dually inoculated plants. But in the
soil-less medium used, with no nutrient limitation,
nutrient acquisition via AM fungus was not significant
for the plant. However, the effect of Bacillus sp.
enhancing intra- and sometimes extraradical fungal
growth and activity resulted in enhancement of the
nutrient content of G. intraradices-colonized plants.
In conclusion, it has been proposed that plants must be
mycorrhizal to thrive in degraded nutrient-poor and arid
soils (Barea 1991, 2000) and that mycorrhizal effects can
be improved by co-inoculation with mycorrhiza-helper
bacteria, which can play an important role in stressed
areas (Requena et al. 1996; Barea 1997). Results from this
present study show that co-inoculation of selected free-
living bacteria isolated from adverse environments and
AM fungi can improve the formation and function of the
AM symbiosis, particularly when the conditions for plants
growth are also adverse. Hence, to restore a self-
sustaining vegetation cover and to combat desertification,
we recommend dual AM fungus-bacterium inoculation.
Acknowledgements This work was financed by UE (Project
ICA4-CT-2001-10057). A.V. is grateful to Fundacin Gran
Mariscal de Ayacucho (Venezuela) and A.M. to ICI (CICYT,
Spain) for their respective research grants.
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