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
The three strains used in this study were identified as P. putida according to FAME analysis Table 1.
None of the strains was producing cyanide Table 1. 3.1. Bacterial impact on early root development
under gnotobiotic conditions The three strains were shown to differ with respect
to their ability to affect the initial root development. Exposure of pea seeds on agar to the three strains
I. Berggren et al. Applied Soil Ecology 16 2001 169–177 173
Table 2 The deleterious impact of Pseudomonas putida strains on development of peas Pisum sativum cv. Capella 3 weeks after inoculation in
gnotobiotic growth pouches
a
Plant part harvested Bacterial treatment cfuml
Å313 AT5
AT8 None
log 3 log 5
log 7 log 3
log 5 log 7
log 3 log 5
log 7 Shoot
120.5 80.3
b
62.7
b
126.3 72.0
b
106.4
b
132.7 123.2
73.0
b
127.9 Root
161.0
b
108.5
b
84.7
b
155.3
b
112.0
b
164.8
b
171.2
b
184.7 93.4
b
193.9
a
Values represent mean dry weight mg per plant, n = 8.
b
Significant at P ≤ 0.05 compared to none bacterial treatment.
resulted in significant P ≤ 0.05 retardation of root growth when compared to the control. Average root
length mm per plant for control was 25.1, Å313 was 7.9, AT5 was 21.2 and for AT8 it was 19.7. Å313 had
the strongest deleterious impact expressed as short, crooked and discoloured roots. In comparison, when
exposed to AT8, in 60 of cases the roots were short and crooked with no discoloration while AT5 caused
no observable negative effect on the roots compared to the non-inoculated controls.
In growth pouches, treatment with AT5 and Å313 lowered the shoot dry weight at the two highest cell
densities used and reduced root dry weight at all three cell densities compared to the control Table 2. In
contrast, strain AT8 reduced shoot and root dry weight only at the highest cell density, log 7 cfuml. Length
measurements were found correlated with two of the root dry weight as a result of Å313 and AT8 treat-
ments, whereas AT5 showed only a growth reduction of the taproots after 21 days at log 5 cfuml Fig. 2.
Treatment with AT5 showed an apparently healthy root system but with visibly thinner and sparser roots.
AT8 at a density of log 7 cfuml, induced shortened taproots, poor lateral root development and occasional
discoloration of the taproot tip. Treatment with Å313 altered root morphology irrespective of cell densities.
At a high cell density, root length was drastically re- duced, also lateral root formation was either inhibited
or absent. Most taproots, and even the lateral roots if present, were discoloured, shrunken and dry. It was
possible to re-isolate all three strains from the roots on TSA at the end of the experiment.
Bacteria-induced root hair deformations were ob- served on root hairs within 3 days after inoculation
Fig. 3. Å313 showed the strongest effect on root hair morphology.
Fig. 2. The deleterious impact of bacterial inoculum densities on root development of peas Pisum sativum cv. Capella in gnoto-
biotic growth pouches 4, 11 and 21 days after inoculation with different cell densities of Pseudomonas putida strains, Å313, AT5
and AT8
s
log 3 cfuml;
h
log 5 cfuml; △ log 7 cfuml;
j
control.
174 I. Berggren et al. Applied Soil Ecology 16 2001 169–177
Fig. 3. Impact of Pseudomonas putida strains on pea root hair development 3 days after inoculation: a control; b strain A313; c strain AT5; d strain AT8.
I. Berggren et al. Applied Soil Ecology 16 2001 169–177 175
Table 3 The deleterious impact of Pseudomonas putida on pea growth in soil 3 weeks after inoculation with log 7 cfuml
Plant part Bacterial treatment
Seed inoculation
a
Seedling inoculation
b
Å313 AT5
AT8 None
Å313 AT5
AT8 None
Shoot 64.1
c
94.2 94.2
98.5 144
96 100
142 Root
208.0
c
408.7 367.9
412.4 308
c
242
c
270 404
a
Values represent mean dry weight mg per plant; one seed per pot, n = 10.
b
Values represent mean dry weight mg per pot; three seeds per pot, n = 5.
c
Significant at P ≤ 0.05 compared none bacterial treatment.
3.2. Bacterial impact on early plant development under non-sterile conditions
Germination was monitored 5 days after the seed inoculation, showing a delay in germination compared
to control 90 whereas the same was 70 for Å313 and 50 for AT5 or AT8. After 2 weeks however, per-
cent germination reached to 90 in all pots irrespec- tive of treatment. Table 3 shows that only the treatment
with Å313 resulted into a significant lower dry matter and root length than uninoculated control plants.
As further shown in Table 3, pre-germinated seeds treated with AT5 or AT8 had negative effects on shoot
and root dry weights though not always significant compared to control plants. However, Å313 showed
its deleterious effect only on roots.
4. Discussion
