170 I. Berggren et al. Applied Soil Ecology 16 2001 169–177
individual strain have been based upon measurements of the length and weight of the plant root and shoot
Nehl et al., 1996. Studies seldom consider the de- velopment of the root system in the presence of DRB.
It is thus important to include morphological andor structural changes in the root system when classifying
DRB, since bacteria-induced alterations may finally have a great impact on plant health.
Detailed understanding of the inhibitory traits of bacteria would lead to the identification of mecha-
nisms underlying the competition and survival be- tween various groups of bacteria in the rhizosphere.
This information will be essential to create the con- ditions necessary to promote the legume-Rhizobium
symbiosis or to favour plant growth promoting rhi- zobacteria PGPR in cropping practices. With this
aim, we investigated whether three previously iso- lated and characterised DRB strains differed in their
deleterious activity on the model plant, pea, under different environmental conditions, before studying
their interrelation with Rhizobium establishment.
2. Materials and methods
2.1. Plant material and growing conditions A white-flowered, semi-leafless and short-stemmed
field pea, Pisum sativum cv. Capella, from Svalöf Weibulls Ltd., Sweden, is a field pea widely grown in
Sweden that was used as model plant. The seeds were fractionated by size and only apparently healthy seeds
with a diameter between 7.0 and 7.5 mm were used.
Plants were grown in growth pouches in the growth chamber with daynight temperature of 1715
◦
C, with a 16 h day length and a mean photosynthetic photon
flux density of 45 klx and 70 humidity. For plants grown in soil, greenhouse conditions were 16–20
◦
C,
Table 1 Some characteristics of the Pseudomonas bacterial strains used in this study
Strain Source
Fluorescence Identity
a
Similarity index HCN production
Deleterious response on plants Å313
Pea roots –
P. putida 0.769
– Wheat, oats, Brassica and spinach
b
AT5 Pea root rot infested soil
+ P. putida
0.662 –
Pea AT8
Pea root rot infested soil +
P. putida 0.754
– Pea
a
Fatty acid methyl ester analysis Sasser, 1990.
b
Åström 1990.
light, if necessary, was supplemented with Philips HPI-T 400 W mercury lamps to give 10 klx at plant
height to ensure 16 h of light period. The soil used originated from a nearby farm, sit-
uated at Ultuna, Sweden, 60
◦
N, 17
◦
E. The soil was a loamy sand, pH 7.2, with a total nitrogen content
of 0.02, an organic matter of 0.4, slightly soluble phosphorus Egnér et al., 1960 9 mg100 g soil and
soluble phosphorus 49 mg100 g soil, and with no pre- vious treatment with fungicides. The soil was moist-
ened to 70 of water holding capacity before planting.
2.2. Bacterial characteristics Three bacterial strains, one non-fluorescent Å313
and two fluorescent AT5 and AT8 were identified as Pseudomonas P. putida using fatty acid methyl
ester analysis FAME, a well-established method for identification Sasser, 1990. Å313, previously shown
to be deleterious, was used as the model organism Åström, 1990. AT5 and AT8 were selected for their
deleterious effects as shown in preliminary tests. The origin and known characteristics of the strains are
presented in Table 1. Stock cultures of the bacterial strains, suspended in sterilised distilled water, were
stored at −70
◦
C prior to use. As inoculum, the bac- terial cell suspensions were prepared by suspending
24 h old cultures grown on TSA Tryptic soy broth agar, 3 gl, Technical agar, 10 gl. Cells were added in
10 mM MgSO
4
to OD 0.3 at 560 nm log 7 cfuml and diluted further, if necessary, in 10 mM MgSO
4
before use.
Cyanide production by the P. putida strains was determined by observing colour changes from yellow
to brown or reddish brown on picrateNa
2
CO
3
paper after 48 h incubation Lorck, 1948. For this, bacterial
suspension was spread on TSA with or without glycine 4.4 gl, two plates for each strain and treatment. The
I. Berggren et al. Applied Soil Ecology 16 2001 169–177 171
papers were fixed to the bottom of a Petri dish lid diameter 90 mm and dish was sealed with parafilm.
Plates left uninoculated and plates inoculated with a known cyanide producing bacterial strain were used
as controls.
2.3. Bacterial impact on early root development under gnotobiotic conditions
2.3.1. Development of roots A initial experiment was carried out for testing
whether the P. putida strains had an impact on the initial pea root development before further studies in
the growth pouches. Seeds were surface-sterilised for 5 min in sodium hypochlorite 0.5 and soaked in
hydrogen peroxide 0.75 for 30 min, followed by incubation in bacterial cell suspensions for 30 min.
Seeds treated with 10 mM MgSO
4
were used as con- trols. Following inoculation, the seeds were placed
on large Petri dishes diameter 190 mm containing water agar 1.5. One Petri dish with 15 seeds was
used for each strain and incubated in dark at 22
◦
C. Root length was measured and root development was
evaluated after 5 days. 2.3.2. Development of root hairs
Pre-germinated pea seedlings were prepared as fol- lows. A glass jar diameter 190 mm, height 90 mm
was filled with 200 g vermiculite and 750 g distilled water, covered with a glass cover and autoclaved
at 121
◦
C for 30 min 1 day before use. Thirty-five surface-sterilised seeds per jar, prepared as above,
were placed 20 mm deep with the notch on the seeds placed in the same position, pointing down into the
vermiculite and allowed to germinate in darkness at 22
◦
C for 4 days prior to use. Microbial contamina- tion of the seedlings was checked on tryptone yeast
extract agar. Only contamination-free seedlings with straight taproots about 3–5 cm long were used in the
experiment.
After pre-germination, the seedlings were inocu- lated with each bacterial suspension log 5 cfuml by
soaking them for 30 min, and transferred to a Fåhraeus slide with one seedling per slide according to So-
masegaran and Hoben 1994 modified so cover slips were glued to the object glasses using 2 mm glass
beads as spacers instead of agar to ensure good root development. The object glasses were 75 mm×25 mm
in size and cover slips were 40 mm × 24 mm. The Fåhraeus slides were placed vertically in test tubes of
diameter 36 mm supplied with 35 ml Fåhraeus solu- tion. Treatments included exposure to Å313, AT5 and
AT8 with six replicates each. Following inoculation, the tubes were kept in a growth chamber and scored
microscopically at intervals for presence of root hair deformations.
2.3.3. Development of root structure Seedlings, prepared as above, were inoculated with
bacterial cell suspensions at three concentrations log 3, 5 and 7 cfuml by soaking them for 30 min.
Cell densities were related to viable cell numbers, measured as cfuml of cell suspension by the standard
dilution plate counting technique. Seedlings incu- bated in 10 mM MgSO
4
were used as controls. Two inoculated seedlings were placed into a sterile growth
pouch, prepared as follows. Plastic growth pouches consisting of polyethylene
bags of size 230 mm × 150 mm × 0.17 mm were pre- pared using a sealing unit. After preparation the bags
were treated at 50
◦
C for 3 days before use to minimise interference with contaminants. A cellulose filter, Mil-
lipore AP10 with a size of 195 mm × 145 mm × 2 mm was saturated in a plant nutrient solution Ols-
son and Alström, 1996 and autoclaved at 121
◦
C for 20 min immediately before use, and inserted asepti-
cally into each pouch. To stabilise the floppy pouch, the cellulose filter was first attached to a sterile glass
plate 195 mm × 145 mm × 2 mm before placing it in the pouch. At the opening of each pouch, two ster-
ile polyethylene funnels were arranged to support the emerging plants. Each funnel was fitted with a ster-
ile cellulose filter Munktell, and a sterile wick of cotton to retain moisture for the emerging seedlings
Fig. 1.
Growth pouches with seedlings were kept at a slight angle in a growth chamber and covered with plastic
film for the first 4 days to prevent from evaporation. Moisture losses in the pouches were compensated for
by injecting sterilised distilled water or nutrient solu- tion aseptically with a sterile syringe at the bottom of
the pouches. The hole thus created was then sealed with sterile tape. The pouches did not contain a free
water level, because the cellulose filter was soaked di- rectly with nutrient solution. Root development was
periodically observed and the position of the tip of the
172 I. Berggren et al. Applied Soil Ecology 16 2001 169–177
Fig. 1. Growth pouch system used to study bacterial im- pact on pea root development: a growth pouch of polyethy-
lene 230 mm × 150 mm × 0.17 mm in size containing a sterilised cellulose filter, Millipore AP10 with a size of
195 mm × 145 mm × 2 mm, saturated with a plant nutrient solu- tion; b sterile polyethylene funnel to support the emerging plant,
containing a sterile cellulose filter Munktell and a sterile wick of cotton; c paper sheet to protect the roots from illumination.
taproot was marked on the pouches at days 0, 4, 11 and 21.
The plants were harvested after 21 days of growth and the plant shoots and roots were dried at 60
◦
C and weighed. At harvest, one pouch with two root
systems from each treatment was randomly removed and homogenised in sterile 10 mM MgSO
4
in order to determine degree of bacterial colonisation and growth.
Serial dilution’s were plated on TSA and incubated over night at 28
◦
C. These plants were not included in the statistical analyses. The experiment was set up in
a random design with nine replicates.
2.4. Bacterial impact on early plant development under non-sterile conditions
When carrying out the greenhouse study, the method used was based on the experimental set up
prepared by Åström and Gerhardson 1988. Pea seeds were rinsed and soaked in 10 mM MgSO
4
at 10
◦
C, for 1 h. The seeds were then inoculated with bacterial suspensions by agitating them for 2 h.
Seeds agitated in 10 mM MgSO
4
only were used as controls. Treated seeds were sown on soil surface in
pots 90 mm width, 110 mm deep containing moist- ened field soil one seed per pot and n = 10 per
strain, covered with a thin layer of soil which was slightly moistened with sterile distilled water. Pots
were placed in a greenhouse and covered with plastic film for the first 2 days to prevent evaporative losses.
Plants were watered as necessary with sterile distilled water to maintain water holding capacity. Emergence
and shoot development were recorded at regular in- tervals. Shoots and roots were harvested after 21 days
of growth, dried separately at 60
◦
C and weighed. Experiment was repeated but with some modifica-
tions, pea seeds were soaked in sterile 10 mM MgSO
4
overnight at 22
◦
C followed by thorough rinsing with sterile 10 mM MgSO
4
and left for germination on sterile moist filter papers for 3 days. Three seedlings,
each having 2–3 cm taproots, were placed in each pot of same size as mentioned above, containing
moistened soil. Following planting, 5 ml of bacterial suspension were poured over each seedling, five repli-
cates per bacterial treatment. Corresponding control plants were treated with a similar amount of sterile
10 mM MgSO
4
. The seedlings were then covered with a thin layer of soil and the surface was slightly moist-
ened with sterile tap water. Rest of the procedure was same as above.
2.5. Statistical analyses The data from all experiments were analysed using
one way ANOVA Newbold, 1991.
3. Results
