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Appl Environ Microbiol, June 1998, p. 2304-2307, Vol. 64, No. 6
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Impact on Arbuscular Mycorrhiza Formation of
Pseudomonas Strains Used as Inoculants for Biocontrol of
Soil-Borne Fungal Plant Pathogens
J. M.
Barea,1,*
G.
Andrade,1
V.
Bianciotto,2
D.
Dowling,3
S.
Lohrke,3
P.
Bonfante,2
F.
O'Gara,3 and
C.
Azcon-Aguilar1
Departamento de Microbiología del
Suelo y Sistemas Simbióticos, Estación Experimental del
Zaidín, Consejo Superior de Investigaciones
Científicas, 18008 Granada, Spain1;
Dipartimento di Biologia Vegetale, Università di
Torino, Turin, 10125, Italy2; and
Microbiology Department, University College, Cork,
Ireland3
Received 19 September 1996/Accepted 25 February 1998
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ABSTRACT |
The arbuscular mycorrhizal symbiosis, a key component of
agroecosystems, was assayed as a rhizosphere biosensor for evaluation of the impact of certain antifungal Pseudomonas inoculants
used to control soil-borne plant pathogens. The following three
Pseudomonas strains were tested: wild-type strain F113,
which produces the antifungal compound 2,4-diacetylphloroglucinol
(DAPG); strain F113G22, a DAPG-negative mutant of F113; and strain
F113(pCU203), a DAPG overproducer. Wild-type strain F113 and mutant
strain F113G22 stimulated both mycelial development from Glomus
mosseae spores germinating in soil and tomato root
colonization. Strain F113(pCU203) did not adversely affect
G. mosseae performance. Mycelial development, but not spore
germination, is sensitive to 10 µM DAPG, a concentration that might
be present in the rhizosphere. The results of scanning electron and
confocal microscopy demonstrated that strain F113 and its
derivatives adhered to G. mosseae spores
independent of the ability to produce DAPG.
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TEXT |
An increasing demand for low-input
agriculture has resulted in greater interest in soil microorganisms
that increase soil fertility or improve plant nutrition and health.
However, in addition to testing the ability of microbial inoculants to
improve plant performance (12), it also is critical to
assess the impact of these inoculants on other key rhizosphere
processes. Because of the importance of arbuscular mycorrhizal (AM)
associations (5), biological control agents must be
compatible with the formation and functioning of AM associations
(6). In this context, it has been found that certain
Pseudomonas strains which produce antimicrobial metabolites
(25) and fungal biocontrol agents, such as
Trichoderma sp. (9) and Gliocladium
sp. (26), do not exhibit inhibitory effects on AM fungi.
The aim of the present study was to explore the impact of biocontrol
Pseudomonas strains which do and do not produce the
antifungal metabolite 2,4-diacetylphloroglucinol (DAPG) on the
formation of AM associations by Glomus mosseae (Nicol.
and Gerd.) Gerdemann and Trappe, a representative AM fungal species
from temperature ecosystems (29). Phloroglucinol antibiotics
are phenolic metabolites with antimicrobial properties (8).
In particular, DAPG is involved in the biocontrol activity of the
plant-growth-promoting rhizobacteria (10, 11, 13, 17). The
parameters measured included AM fungal spore germination (both in vitro
and in soil), the amount of plant-independent mycelial growth (in
vitro), and the degree of establishment of AM associations on the
developing root systems of tomato plants (in soil). In addition, the
interaction between Pseudomonas strains and G. mosseae was examined by scanning electron and confocal microscopy
to determine whether direct cell-to-cell contact or production of DAPG
is a factor in the interaction between the fungus and the bacteria.
Microbial strains.
The isolate of G. mosseae used
in this study was obtained originally from Rothamsted, United Kingdom.
Wild-type Pseudomonas sp. strain F113 was isolated from the
rhizosphere of mature sugar beets (13). The following two
genetically modified derivatives of strain F113 also were tested:
F113G22, a DAPG-negative mutant of F113, which was constructed by using
Tn5::lacZY (28); and Pseudomonas sp. strain F113(pCU203), a DAPG overproducer
(13). The antibiotic resistance characteristics of these
Pseudomonas strains are as follows: F113, 100 µg of
rifampin per ml; F113G22, 50 µg of kanamycin per ml; and
F113(pCU203), 200 µg of chloramphenicol per ml. The latter two
strains are not resistant to rifampin.
Mycorrhizal fungal spore germination and mycelial growth in
vitro.
The experiments to examine mycorrhizal fungal spore
germination and mycelial growth in vitro were conducted as described by Azcón-Aguilar et al. (4). Sporocarps of G. mosseae were obtained from rhizospheres of onion (Allium
cepa L.) plants grown in pot cultures. The rhizosphere samples
were kept in polyethylene bags at 4°C, and after collection the
sporocarps were stored on damp filter paper at 4°C. Resting spores
freshly excised from the sporocarps were surface sterilized in a
solution containing 20 g of chloramine T per liter, 200 mg of
streptomycin per liter, and 1 drop of Tween 80 per liter
(24) for 20 min and were then washed five times in sterile
water. The Pseudomonas biocontrol strains F113 and F113(pCU203) (antifungal strains) and the mutant strain F113G22 (with impaired biocontrol ability) were grown at 28°C for 24 h on Luria-Bertani (LB) medium (10 g of tryptone per liter, 5 g of
yeast extract per liter, 5 g of NaCl per liter, 15 g of Difco agar per liter), centrifuged, and then washed three times in 0.25× Ringer's solution (Oxoid) prior to use.
Pseudomonas suspensions were adjusted to an optical density
at 650 nm of 0.4, which corresponded to a concentration of
108 CFU ml
1, and 50-µl portions were spread
onto the agar surfaces in petri dishes (diameter, 9 cm) containing
water agar (0.8% Bacto Agar [Difco]) buffered with 10 mM MES
[2-(N-morpholino)ethanesulfonic acid] (9). The
final pH after sterilization at 120°C for 20 min was 7.0. Six
surface-sterilized spores of G. mosseae were transferred
individually to each Pseudomonas-inoculated petri dish;
these six spores were located at the vertices of an imaginary hexagon
with sides approximately 3.5 cm long. Incubation was at 25°C in the
dark, and the plates were sealed with Parafilm. Each treatment
consisted of five replicate plates and five control plates containing
G. mosseae spores growing axenically. Spore germination was
evaluated after 28 days. A spore was considered germinated if a germ
tube was clearly visible. Hyphal growth from germinated resting spores
also was assessed by light microscopy. Mycelial growth was estimated by
a gridline intersect method (4).
In another experiment, the effect of the antimicrobial metabolite DAPG
on AM fungal spore germination and mycelial growth
was tested after
purified DAPG was added to axenic cultures of
G. mosseae
spores. Pure DAPG was obtained from the Chemistry Department,
University College, Cork, Ireland. A stock DAPG solution (100
mg
ml
1 in methanol) was prepared and filter sterilized
(Millipore).
Appropriate dilutions were mixed with melted water agar to
obtain
concentrations ranging from 1 to 1,000 µM.
To examine the effects of the
Pseudomonas strains on AM
fungal spore germination in soil, the following experiments, based
on
previous studies, were carried out (
18,
25). Each
experimental
unit consisted of a slide frame that held two membrane
filters
(diameter, 45 mm; pore size, 0.45 µm; type HT Tuffryn).
Twenty
unsterilized
G. mosseae spores were introduced
between the two
membranes. Twenty-five grams of soil was placed in a
9-cm-diameter
petri dish, and the sandwich units were then laid onto
this soil
layer. An agricultural soil collected from Granada Province
(Spain)
was used. The characteristics of this test soil, a Cambisol,
were
as follows: pH (H
2O), 6.8; concentration of available
(NaHCO
3-extractable)
P, 15 mg liter
1; total N
concentration, 2,600 mg liter
1; organic C concentration,
0.8%. The soil consisted of 58.7% sand,
26.4% silt, and 14.9% clay.
This soil was collected from the upper
20 cm of the soil profile, and
the soil was sieved through a 2-mm
mesh prior to use. One milliliter of
a bacterial suspension containing
10
8 CFU ml
1
(as determined by optical density) or 1 ml of a DAPG dilution
(range of
concentrations used, 1 to 1,000 µM) was applied to the
membranes.
Another 25 g of soil was added to cover the sandwich
units. The
soil was moistened with distilled water to field capacity,
and the
petri dishes were sealed with Parafilm. The dishes (five
replicates for
each treatment) were then incubated at 25°C for
2 weeks. Upon removal
from each dish the sandwiches were opened,
and the germ tubes were
stained with trypan blue (
27). The percentage
of germinated
spores was calculated for each treatment. All experiments
were
conducted twice, and the data for the level of germination
for each
treatment, including both in vitro and soil tests, were
subjected to a
hypothesis test against the corresponding control
data. The z statistic
for germination tests was calculated from
the experimental data and
compared with the z statistic tabulated
at the 5% significance level.
Mycelial growth data were processed
by the analysis of variance method
and Duncan's multirange test
(
P 
0.05).
Results summarized in Table
1 showed that
none of the
Pseudomonas strains tested inhibited germination
or mycelial development
of
G. mosseae in vitro or in soil.
Moreover, strains F113 and
F113G22 had a significant stimulatory effect
on mycelial growth.
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TABLE 1.
Effects of Pseudomonas strains on G. mosseae spore germination in vitro and in soil and on
mycelial growth in vitro
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The effects of purified DAPG on spore germination and on the subsequent
development of the fungal mycelium are shown in Table
2. These results indicate that only the
highest concentration
of the antifungal compound assayed (1,000 µM)
inhibited spore
germination. Development of fungal mycelia was more
sensitive,
and significant inhibition was observed in the presence of
10
µM DAPG.
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TABLE 2.
Effects of the antifungal compound DAPG on G. mosseae spore germination in vitro and in soil and on mycelial
growth in vitro
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Attachment of bacteria to spores.
Sporocarps of G. mosseae were surface sterilized by treating them with a solution
containing 4% chloramine T and 300 µg of streptomycin per ml for 30 min and were rinsed five times over a 1-h period with sterile distilled
water. A portion of each sporocarp was manipulated with thin pointed
forceps to remove the finely branched and interconnected hyphae
surrounding the spores. The single spores were then sterilized as
described above and were cleaned by subjecting them to four 30-s
sonication pulses followed by four rinses with sterile distilled water.
The
Pseudomonas strains were grown separately in LB liquid
medium overnight at 28°C with gentle shaking. After centrifugation
at
3,000 ×
g for 20 min, each supernatant was discarded,
and the
pellet was resuspended in 15 ml of 50 mM phosphate buffer (pH
7.2). The concentration of bacterial cells in each suspension
was
between 10
7 and 10
8 CFU ml
1 and
was adjusted by using optical density measurements.
For the attachment assay, spores were prepared and examined by scanning
electron and confocal microscopy as previously described
by Bianciotto
et al. (
7).
When the three
Pseudomonas strains were incubated with
sporocarps and isolated spores of
G. mosseae, they all
displayed the
same attachment behavior with these fungal structures.
Spores
treated with
P. fluorescens F113, F113G22, or
F113(pCU203) had
only a few bacterial cells on their cell walls.
When the sporocarps
were incubated with strain F113, a few bacterial
cells were found
among the hyphal network that covers the spores.
Identical results
were obtained with strain F113G22. To rule out the
possibility
that the fungal surfaces were contaminated previously by
other
bacteria, some spores of
G. mosseae were incubated
with no bacteria,
and the fungal surfaces were always free of bacterial
cells. These
results are consistent with those reported previously by
Bianciotto
et al. (
7), who found that bacterial strains
always adhere
to
Gigaspora margarita, another AM
fungus.
Mycorrhiza formation.
Five-day-old seedlings of tomato
(Lycopersicon esculentum L.) obtained from
surface-sterilized (50% [vol/vol] commercial bleach solution, 10 min) seeds germinated on wet filter paper in petri dishes were
transplanted into pots (capacity, 1 liter) containing Granada
agricultural soil (described above) that had been sieved through a
4-mm-mesh screen.
The mycorrhizal fungal inoculum was obtained in a pot culture
(
1) with
A. cepa L. (onion) as the host plant and
was added
to produce a rhizosphere soil containing five sporocarps per
g
(with an average of six mature spores per sporocarp) together
with
some single spores, mycelium, and mycorrhizal root fragments.
Fifty
grams of this mycorrhizal inoculum per pot was thoroughly
mixed with
the soil. The
Pseudomonas cultures tested were
grown
on LB medium, washed three times in 0.25× Ringer's solution
(Oxoid),
and then adjusted to an optical density at 650 nm of 0.4. Two
milliliters per seedling (one seedling per pot) was applied to
the
roots at the time of transplantation.
The test plants (five replicates per treatment) were grown in a
greenhouse with a day-night cycle consisting of 16 h of light
at
21°C and 8 h of darkness at 15°C at a relative humidity of
50% and a photosynthetic photon flux density of 600 to 700 mmol
m
2 s
1. During the assay, the plants were
fertilized (10 ml/week/pot)
with Long Ashton nutrient solution
(
19) lacking P. Throughout
the experiment, the pots were
weighed every day, and the water
loss was compensated for by top
watering (with tap water) to reach
field capacity. The pot bioassay was
carried out twice with two
different growth periods (6 and 8 weeks).
When the plants were
harvested, plant shoot and root dry weights were
recorded after
drying at 70°C. Representative root samples were
stained for mycorrhiza
examination (
30). The percentage of
the total root length that
became mycorrhizal was calculated by a
gridline intersect technique
(
16).
At the end of the experiment and before biomass and AM quantifications
were performed, representative root samples from seedlings
in each pot
were suspended in 9 ml of Ringer's solution and vortexed
for 7 min,
and serial dilutions were inoculated onto LB medium
containing the
appropriate antibiotics to measure colonization
of the tomato
rhizosphere by the
Pseudomonas inoculants. Data
from the pot
experiments were processed by the analysis of variance
method and
Duncan's test (
P 0.05). In the case of the
percentage
of mycorrhizal root length, the data were subjected to
arcsine
square root transformation. To evaluate bacterial population
density,
the data were transformed to a log scale prior to statistic
analyses.
Table
3 summarizes the impact of the
Pseudomonas strains on the formation of AM associations.
Pseudomonas inoculation increased
the percentage of root
length that became mycorrhizal (significantly
for F113 and F113G22) and
improved shoot growth in the mycorrhizal
plants by the second harvest
time tested (8 weeks). Log-transformed
data showed that there
were no statistically significant differences
(
P = 0.05) in the level of establishment in the tomato rhizosphere
among
the three strains. The log values were 7.55, 7.75, and 7.44
CFU g
of dry roots
1 for strains F113, F113G22, and F113(pCU
203), respectively. These
population data were obtained after 6 weeks of plant growth.
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TABLE 3.
Effects of Pseudomonas strains on mycorrhiza
formation and biomass production by tomato plants growing for
either 6 or 8 weeks in soil microcosms in the greenhouse
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Discussion and conclusions.
Data presented in this paper
indicate that certain Pseudomonas strains that produce DAPG
and that are used as biocontrol agents (11) do not exhibit
detrimental effects on the AM fungus G. mosseae. This
general conclusion is based on the following two facts: (i) no
bacterial treatment adversely affected mycorrhizal colonization (Table
3), and (ii) even with the DAPG overproducer F113(pCU203) the
mycelial development (as determined by germination tests) was not
significantly less than the mycelial development when no bacteria were
introduced (Table 1).
In spite of the fact that the rhizosphere is nutritionally enriched
compared to bulk soil, which may affect synthesis of DAPG
(
28), the data from the germination test performed in
relatively
nutrient-poor bulk soil and the data for the development of
AM
associations in the rhizosphere revealed similar tendencies, with
the wild-type strains F113 and F113G22 stimulating both mycelial
development and colonization of roots and the DAPG overproducer
strain
F113(pCU203) producing effects similar to those of the
control. It
is likely that F113(pCU203) produced the antifungal
compound during
the experiment but at doses which do not have
an adverse effect
compared to the control but are rather inhibitory
compared to the other
two strains. The concentration of the antifungal
compound DAPG required
to produce negative effects on the germination
and development of AM
fungal spores (10 µM) might be present in
the rhizosphere of
Pseudomonas-inoculated plants (
8).
Water agar is a nutrient-poor medium, and this may have affected DAPG
synthesis by the strains in this study. The data in
Table
1, which show
that F113(pCU203), a DAPG overproducer, inhibited
mycelial
development compared to the other two strains, suggest
that DAPG
production could have occurred. However, we do not support
using
antagonism on water agar as a method for looking at DAPG
production.
Previously, other authors (
20,
23) have described the
resistance of various crop plants and microorganisms to DAPG. The
results indicate that the levels of DAPG required to inhibit fungal
pathogens in vitro may also result in phytotoxic effects
(
31).
A comparison of the levels of DAPG which result in
50% inhibition
of fungal pathogens (
23) with the levels
that result in significant
inhibition of
G. mosseae
suggested that AM fungi are at least
as sensitive as pathogenic fungi.
Therefore, it appears that intrinsically
greater resistance to DAPG may
not account for the observed lack
of a negative effect of biocontrol
strains on
G. mosseae (in soil).
Another interesting finding is that F113 and F113G22 are able to
improve the formation of AM associations, as previously shown
for other
rhizosphere microorganisms (
3). The precise mechanism(s)
that accounts for such microbial stimulation, however, has not
been
clearly identified yet. Most current evidence indicates that
many
microorganisms develop functions in the rhizosphere which
may affect
not only the plants but also other microbial members
of the soil
community (
21,
22). This is an effect exhibited
by the
so-called "mycorrhiza helper bacteria," which have a positive
influence on formation of ectomycorrhizal (
14,
15) and AM
(
3) associations. Specialized activities, such as the
production
of vitamins, amino acids, hormones, etc., may be operating
in
microbe-microbe interactions involving AM fungi and
Pseudomonas strains (
2) and may account for the
stimulatory effects found
in this study. On the basis of these
observations and due to the
importance of AM associations in
agroecosystems, the release of
microbial inoculants that produce
antifungal metabolites deserves
a detailed analysis to determine the
possible effects on the performance
of AM fungi (
6).
In summary, it is significant that the biocontrol organism
Pseudomonas sp. strain F113 did not exhibit antifungal
activity
against
G. mosseae, a representative mycorrhizal
fungus, and that,
in addition, this strain had a significant
stimulatory effect
on mycelial development from
G. mosseae
spores and on the overall
processes involved in the formation of AM
associations in soil.
This stimulation did not appear to be related to
DAPG production,
as strain F113G22, which is deficient in DAPG
production, also
had a such stimulatory effect.
The lack of any inhibitory activity by the biocontrol agent
Pseudomonas sp. strain F113 against a beneficial fungal
symbiont
(
G. mosseae) and the lack of inhibitory activity by
other
Pseudomonas strains (
25) have implications
concerning the evaluation of
biocontrol strains not only with regard to
target fungal pathogens
but also with regard to the ecological impact
of biocontrol strains
on beneficial resident soil microbial
populations.
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ACKNOWLEDGMENTS |
This study was supported by the EU Biotechnology Programme (IMPACT
projects BIO2-CT93-0053 and BIO4-CT96-0027).
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FOOTNOTES |
*
Corresponding author. Mailing address: Departamento de
Microbiología del Suelo y Sistemas Simbióticos,
Estación Experimental del Zaidín, CSIC, c/Prof. Albareda
1, Granada 18008, Spain. Phone: 34 9 58 121011. Fax: 34 9 58 129600. E-mail: jmbarea{at}eez.csic.es.
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Appl Environ Microbiol, June 1998, p. 2304-2307, Vol. 64, No. 6
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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