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Soil Biology & Biochemistry 32 (2000) 1443±1451
www.elsevier.com/locate/soilbio

Characterisation of bacteria in soils under barley monoculture
and crop rotation
Stig Olsson*, Sadhna AlstroÈm
Plant Pathology, Department of Ecology and Crop Production Science, Swedish University of Agricultural Sciences, Box 7043, S-75007 Uppsala,
Sweden
Received 28 September 1999; received in revised form 26 January 2000; accepted 14 February 2000

Abstract
Rhizobacterial populations on barley roots, originating from experimental ®elds with barley monoculture (MC) and crop
rotation (CR), were analyzed for their fatty pro®les. In the ®rst part of the study, the pro®les of 1188 isolates were statistically
analyzed to identify clusters of bacteria with a possible high prevalence in MC or CR soil. One such cluster was found, termed
Ps4-C4, with characteristically high contents of C12:1 3OH (9.8%) and an unidenti®ed fatty acid with the equivalent chain
length (ECL) of 12.35, which was provisionally named ECL12.35 (4.5%). Bacteria in Ps4-C4 were also rich in C10:0 3OH
(8.3%) and C12:0 3OH (8.1%). The cluster consisted of 109 isolates, 86 from MC populations and 23 from CR, none of which
could be identi®ed by means of fatty acid analysis. In the second part of the study, fatty acid pro®les of 240 microbial
populations from the same ®elds were analyzed. Results showed higher relative contents of C12:1 3OH, ECL12.35, C10:0 3OH
and C12:0 3OH in MC populations, which also had a high proportion of C12:0 2OH and C16:0. A detailed statistical analysis
of the correlations between the fatty acids indicated that Ps4-C4 alone explained the higher portions of C12:1 3OH and

ECL12.35 in populations from MC soil, while other bacterial groups are seem to have contributed to the elevated contents of
C10:0 3OH and C12:0 3OH. Some common functional characteristics of bacteria in the Ps4-C4 cluster are also described. 7 2000
Elsevier Science Ltd. All rights reserved.
Keywords: Rhizosphere bacteria; Barley; Monoculture; Hydroxy fatty acids

1. Introduction
The interface between soil and plant roots, the rhizosphere, is a dynamic habitat. In the surrounding
bulk soil, the growth and proliferation of microbes is
normally limited by a shortage of carbon and energy
while the continuous release of organic nutrients in
plant rhizosphere facilitates the activity and multiplication of a large variety of microorganisms. Di€erent
plant species release di€erent organic compounds, and
thus they can encourage a di€erentiated rate of proliferation of the microbiota (Curl and Truelove, 1986;

* Corresponding author. Tel.: +46-18-67-28-63; fax: +46-18-6728-90.
E-mail address: Stig.Olsson@evp.slu.se (S. Olsson).

Grayston et al., 1998). A consequence of this would be
that crop rotation practices in agriculture in the long
run, would in¯uence the composition of the microbial

population in the bulk soil. There are, however, only a
few studies which support this assumption (e.g., Zelles
et al., 1992, 1995). The scarcity of supporting evidence
is partly due to methodological diculties faced by
many researchers while carrying out such investigations.
When studying microbial communities in soil or
in the rhizosphere, researchers choose either to
work with individual isolates or with microbial
communities, both of which strategies have their
merits and drawbacks. An important advantage of
working at the isolate level is that it o€ers possibility of making a detailed description of the members of the communities. At the same time, a major

0038-0717/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved.
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S. Olsson, S. AlstroÈm / Soil Biology & Biochemistry 32 (2000) 1443±1451

drawback is that huge numbers of isolates are

required to characterize and compare compositions
of di€erent communities. Similarly, an important
advantage of working at the community level is
that it may be easier to detect di€erences between
communities but it can then be dicult to interpret
them in biological terms. A third approach can be
to combine the two strategies. Such a combined
approach was found successful when using fatty
acid methyl ester (FAME) pro®les of bacteria to
identify the di€erences between the composition of
bacterial populations in bulk soil and in the rhizosphere (Olsson et al., 1999; Olsson and Persson,
1999).
In studies where microbial communities are
characterized by means of fatty acid pro®ling, they
are usually based on extraction of phospholipids
and/or lipopolysaccharides directly from the soil
samples (Zelles et al., 1992, 1994, 1995, BoÈrjesson
et al., 1998; Steinberger et al., 1999). This direct
lipid extraction represents ®ngerprints of the in situ
microbial soil populations. As an alternative

approach, Olsson and Persson (1999) analysed the
FAME pro®les of populations cultured on a bacterial nutrient medium. Obviously, there is a risk in
the latter approach of either losing information
from the bacteria which are not cultivable and/or in
some other way in¯uencing the in situ composition
of the community by culturing. However, extracting
lipids directly from any soil involves problems in
interpreting the results. Some fatty acids can easily
be used as signatures for certain broad groups of
organisms (Zelles, 1997), but the diculty arises
when discriminating between closely related species.
To be able to describe the fatty acid pro®le of a
microorganism, it has to be cultured and it is likely
that its ``in vitro pro®le'' in some respects di€ers
from its ``in situ pro®le'' in soil. Therefore, it
should be an advantage to analyze cultured communities provided that a relevant database of the
organism's ``in vitro pro®les'' is available.
In the present study, we have implemented further
our earlier combined approach of analyzing FAME
pro®les as biomarkers of bacterial communities

together with those of individual isolates, as in Olsson
et al. (1999) and Olsson and Persson (1999). The principal aim was to ®nd and interpret di€erences between
rhizosphere microbial populations as an e€ect of longterm management practices due to di€erent crop rotations. More speci®cally, we compared long-term barley monoculture (MC) with a more diverse crop
rotation (CR). Attempts were also made to study certain phenotypic characteristics of the bacterial populations speci®c to monoculture soils.

2. Materials and methods

2.1. Experimental ®elds
Soil or plant roots were sampled from three experimental ®elds with long-term crop rotations. The ®elds
are situated at Ultuna, LoÈvsta and SaÈby in the central
part of Sweden. The ®eld experiments at Ultuna were
initiated in 1959 and include plots with barley in
monoculture (MC) and with an eight-year crop rotation having a sequence of: fallow, winter rape, winter
wheat, peas, spring barley, ley, ley and oats (CR). The
crop rotation plots with winter rape and barley had
been treated with manure corresponding to 30 and 20
t haÿ1, respectively.
At SaÈby and LoÈvsta the rotational experiments were
started in 1967 and 1968 and at both the places there
have been treatments with barley in monoculture and

a six-year crop rotation sequence: fallow, winter rape,
winter wheat, oats, spring barley and spring wheat. No
manure has been applied to these ®elds.
At Ultuna and LoÈvsta the clay content of the soil is
40±45% and the pH 6.2. At SaÈby the soil is silt loam
(clay content 17%) with pH 6.1. The barley grain
yields at the three ®elds have been about 5 t haÿ1 in
the rotational plots during the last 15 years. The
monoculture plots yield on an average 10% less than
the rotational plots (Olsson, 1995).

2.2. Sampling bacterial isolates
The method used for sampling bacterial isolates
from the experimental ®elds is presented in detail in
(Olsson et al., 1999; notice, however, that in this paper
the amount of agar has been erroneously written as 10
g lÿ1; the correct ®gure is 15 g lÿ1). Brie¯y, soil
samples were collected from the ®elds on two occasions, in September 1995 and May 1996, and they
were used as a medium for cultivating the bait crop,
barley, under standardized conditions. The roots of

the bait crop were harvested, cleansed from soil
crumbs and macerated in sterile 0.01 M MgSO4. The
suspensions were further diluted in the same medium
and plated on tryptic soy broth agar (TSA15, containing 15 g lÿ1 TSB (DIFCO) and 15 g lÿ1 Oxoid technical agar). After incubating for 4±5 days at 118C, the
bacterial isolates were sampled, approximately 15 isolates per plate. We tried to obtain samples that
re¯ected the composition of each plate with respect to
visual characteristics such as size, color and colony
shape. Bacteria were thus collected from a total of 72
soil-root samples i.e. 2 occasions  3 ®elds  2 crop rotations  2 replicated plots x 3 replicated samples per
plot, which yielded a total of 1188 bacterial isolates.

S. Olsson, S. AlstroÈm / Soil Biology & Biochemistry 32 (2000) 1443±1451

2.3. Sampling bacterial populations
Soil-borne bacterial populations were obtained on
two occasions in June 1999 with a two-week interval,
when the barley plants had developed 2±3 and 4±5
leaves, respectively. The sampling procedure was as
follows: ®ve separate samples were taken from each
plot, each sample consisting of 10±15 barley plants

plus roots and adhering soil. One sample covered 10
cm of a sowing row and they were taken with intervals
of approximately 3 m between the samples. The plants
were dug up with a small spade down to a depth of
10±15 cm and brought to the laboratory. The roots
were mechanically cleansed from loosely adhering soil
crumbs but were not rinsed with water. Approximately
0.8 g fresh weight of barley root with rhizosphere
(rhizo-) soil from each sample was shaken in 10 ml
sterile tap water in a test-tube and left at 68C overnight. The suspensions thus obtained were spread on
TSA5 (0.4 ml per plate, 5 g lÿ1 TSB (DIFCO) and 15
g lÿ1 Oxoid technical agar) aseptically and left for incubation at 88C for 5 days. The bacterial populations
were harvested by sweeping the surfaces of the rotating
agar plates with a plastic loop so as to obtain 100±150
mg fresh weight of bacterial biomass per plate for
FAME analysis (see below for procedure).
Furthermore, on the ®rst sampling occasion, the
populations from bulk soil were also included for comparison with that of rhizo-soil. For this, along with
each sample taken from the ®elds for rhizo-soil, 2 g
fresh weight of bulk soil was suspended in 10 ml sterile

tap water in a test tube and vigorously vibrated in a
Vortex. These bulk-soil suspensions were spread in the
same way as the root-rhizo-soil suspensions above. On
the second sampling occasion, the e€ect of inoculum
density on population composition was also studied.
For this, the start suspensions were diluted 10-fold and
both the diluted and start suspensions were spread on
TSA5 and subjected to FAME analysis.
In total, 240 microbial populations were thus analyzed from 120 soil-root samples collected on two occasions (barley baitplants with 2±3 and 4±5 leaves,
respectively)  3 experimental ®elds (LoÈvstad, Ultuna
and SaÈby)  2 crop rotations (MC and CR)  2 plots
(each crop rotation was represented by two replicated
plots in each experimental ®eld)  5 replicated samples
per plot.
2.4. Incubation conditions, fatty acid methyl ester
analysis procedure
For FAME analysis of the 1188 isolates, they were
grown on TSA20 containing 20 g lÿ1 TSB and incubated for 24 h at 248C. In contrast, the bacterial populations were cultured on TSA5 before incubation for 5
days at 88C. These modi®cations were considered

1445

necessary because (1) for culturing the single isolates,
we had to follow the standards of Microbial Identi®cation System (MIS, Microbial ID, US) to rely on
their identi®cation, whereas (2) for culturing the populations, we had to be close to soil conditions normally
prevailing in sampled ®elds i.e. low nutrient level and
low temperature regimes.
Results of interest from FAME analysis of single
isolates were con®rmed by reanalyzing some of them
when incubated under the same conditions as the
populations i.e. they were cultured on TSA5 for 5 days
at 88C before analysis. The same isolates were also
incubated according to the MIS standard i.e. 30 g TSB
and incubated for 24 h at 288C to further con®rm
their identity.
FAMEs from the isolates or from the bacterial
populations were extracted according to Sasser (1990).
They were separated on a Hewlett Packard 5890 Series
II gas chromatograph, with a 25 m  0.2 mm methyl
silicone fused silica capillary column, using hydrogen

as the carrier gas. In general, it was possible to identify
individual FAMEs using the peak-naming table component provided by the MIS. When the isolates were
analyzed, some new peaks appeared which could not
be identi®ed by MIS software. Therefore, for the purpose of comparison in this study, these peaks were
given provisional names by referring to their equivalent chain length (ECL), and their peak areas were also
included while calculating the total named FAME
peak area. The relative quantities of individual FAME
peaks were expressed as percentages of the thusde®ned total named FAME peak area.
2.5. Bacterial tests
To determine a possible common pattern in functional characteristics of isolates found representative of
a particular group, they were studied in several ways.
For production of ¯uorescent pigments, all bacteria
were cultured overnight on King's B medium (KBA,
King et al., 1954) and thereafter observed for ¯uorescence under UV light.
Isolates were further tested with respect to several
phenotypes. Their ability to produce proteolytic
enzymes was qualitatively analyzed by growing bacteria on skimmed milk agar (5 g fat-less skimmed milk
and 5 g Oxoid technical agar in 300 ml distilled water
and autoclaving at 1218C for 20 min) and observed for
clear zones around the growing colonies. Assay for cellulolytic activity was done on cellulase indicator agar
plates. This was done by culturing bacteria on TSA20
supplemented with Na-carboxymethylcellulose (5 g lÿ1)
at 258C. Bacterial cultures were ¯ooded with 0.1%
Congo red stain prepared in distilled water for 15 min.
De-staining was carried out thereafter by re-¯ooding
with 1 M NaCl for 15 min. The presence of yellow

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S. Olsson, S. AlstroÈm / Soil Biology & Biochemistry 32 (2000) 1443±1451

halo zones indicated production of cellulolytic
enzymes.
In addition, phenol oxidation was tested by culturing bacteria on gallic acid agar as described in Kloepper et al (1991). Presence of visible lawns after 7±14
days was interpreted as a positive reaction. Whether
they could be recognized by the plant cells was interpreted by their ability to induce hypersensitive reaction
(HR; Klement, 1963). HR was studied on freshly cultured bacteria suspended in sterile 0.1 M MgSO4 to
obtain a concentration of approx. 109 viable cells
mlÿ1after in®ltrating them in leaves of young broad
bean plants. In our experience, the isolates expressing
HR in tobacco leaves also do so in broad bean leaves.
Development of necrosis in the in®ltrated zones within
24±48 h was considered a positive reaction. A positive
bacterial control was included in all the above tests.
Ability to produce a volatile metabolite, hydrogen cyanide (HCN), was measured qualitatively as described
in AlstroÈm and Burns (1989). Change in colour of indicator paper from yellow to brown revealed the presence of HCN.
The biological activity of isolates was evaluated in
terms of their ability to inhibit growth of fungal pathogens. For this purpose, two pathogens, Rhizoctonia
solani KuÈhn and Bipolaris sorokiniana (Sacc.) Shoemaker were selected, cultures of which were obtained
from our own culture collection. This was done by
using dual culture assay on potato dextrose agar
(PDA). A piece of freshly cultured fungus was inoculated in the center of a PDA plate and bacteria were
inoculated equidistantly from the fungus. After incubation for 7±14 days depending on the fungus, growth
of each pathogen under the in¯uence of each isolate
was compared with its growth in control plates lacking
any bacteria.
2.6. Statistical analysis
The variables used in the statistical analysis of the
bacterial isolates and the bacterial populations were
the relative quantities of all named fatty acids throughout. Thus, the ®rst part of the analysis was done with
FAME pro®les of the 1188 bacterial isolates. The aim
was to detect any signi®cant di€erences between isolates originating from MC and CR soil. This analysis
was performed in three partly iterative steps. In the
®rst step, the most important variance components
were summed up by making a principal component
analysis over the dominating fatty acids, i.e., those
acids, the sum of which constitute more than 90% of
the total lipid content for at least 75% of the isolates.
The principal components were used as variables in
the second step to ®nd if there were any indications of
an over-all di€erence between bacteria from MC and
CR soil. If the tests in step 2 were positive, i.e., if the

di€erences were statistically signi®cant, then the
speci®c FAME pro®les and/or the groups of bacteria
of interest were analyzed in detail as step 3.
The second part of the analysis was done on FAME
pro®les of microbial populations so as to evaluate the
results obtained in steps 2 and 3 of the ®rst part.
An earlier analysis of the FAME pro®les of 1188
bacterial isolates revealed that they could be divided
into two main groups: Group 1, with bacteria whose
lipid content consisted mainly (90%) of fatty acids
with unbranched and even-numbered carbon chains,
and Group 2, those with a high content (70%) of
branched and odd-numbered carbon chains (Olsson et
al., 1999). When using MIS (Library TSBA, Version
3.9), which identify bacterial isolates by comparing the
recorded fatty acid pro®les with a database, it was
found that Pseudomonas was the dominant genus in
Group 1, and Cytophaga and Gram positives in Group
2.

3. Results
In the present study, the two bacterial groups,
Group 1 and Group 2, were further analyzed separately. Analysis of Group 1, which consisted of 720 isolates (314 from CR and 406 from MC soil), showed
that 90% of the isolates in this group had >90% of
their total lipid content in ten fatty acids: C15:0 ISO
2OH&C16:1, C16:0, C18:1, C17:0 CYCLO, C12:0,
C12:0 2OH, C10:0 3OH, C12:0 3OH, C12:1 3OH and
an unidenti®ed peak tentatively called ECL12.35. The
fatty acid, C15:0 ISO 2OH, could not be di€erentiated
from C16:1 and therefore these were merged to give
one value, i.e., C15:0 ISO 2OH&C16:1. A principal
component analysis over these fatty acids showed that
the ®rst ®ve components represented 42, 23 14, 9 and
5%, respectively, of the total variation between the isolates.
The overall e€ect of the rotational treatments, MC
or CR, on the FAME pro®les was evaluated by using
the ®rst ®ve principal components as independent variables in a linear regression analysis. This evaluation
showed a statistical signi®cance (for the model: P <
0.0001 and for the PCs < 0.001, 0.09, 0.76, 0.005 and
0 and 2nd component > 2
Ps3: 1st component > 1 and 2nd component < 2
Ps4: 1st component < ÿ2
Ps5: ÿ2 < 1st component < 0 and 2nd component
> ÿ1
Secondly, the validity of these subgroups was corroborated in a cluster analysis over the ®rst ®ve principal
components (Wards linkage method of minimum var-

Fig. 1. Dispersal of 720 isolates in Group 1 into ®ve smaller clusters over the ®rst two principal components.

1448

S. Olsson, S. AlstroÈm / Soil Biology & Biochemistry 32 (2000) 1443±1451

Table 2
Numbers of isolates found in ®ve subgroups by means of interactive
plotting over the ®rst two principal components (Ps1±Ps5) and by
cluster analysis (C1±C5) over the ®rst ®ve components (Ward minimum variance method and Euclidean distances)

C1
C2
C3
C4
C5

Ps1

Ps2

Ps3

Ps4

Ps5

343
0
17
0
37

17
56
34
0
2

28
0
34
0
0

0
0
0
109
12

28
0
1
0
2

iance and Euclidean distances). The correspondence
between the two methods (Table 2) indicated that the
bacteria belonging to both Ps1 and cluster 1, C1 (343
isolates) and those in both Ps4 and C4 (109 isolates)
formed two well-separated clusters. Ps1±C1 cluster
contained 170 isolates from CR compared to 173 from
MC soil, while the distribution was 23 and 86, respectively, in Ps4±C4 cluster. MIS identi®ed 322 of the 343
isolates forming Ps1±C1 as Pseudomonas (128 as P.
putida; 94 as P. chlororaphis; 51 as P. ¯uorescens and
the rest belonging to less common species) with similarity index (SI) > 0.6 whereas all the 109 isolates
forming Ps4±C4 remained unidenti®ed (SI < 0.6).
The mean values and the standard deviations for the
fatty acids in the two bacterial clusters of Group 1 are
shown in Table 3. This table also includes data from
the bacteria in Group 2 and it indicates that the microbial populations with a high frequency of Ps4±C4
can be recognized by high proportions of C12:1 3OH,
ECL12.35, C10:0 3OH and C12:0 3OH.

3.2. Bacterial populations
The groupings presented above were further evaluated by analyses of bacterial populations, which were
obtained from roots of barley plants sampled directly
from the experimental ®elds. The results are summarized in Table 4. All four fatty acids speci®ed above as
characteristic to Ps4±C4 were also found in signi®cantly higher proportions in MC populations from
roots with rhizo-soil than in corresponding CR populations.
The correlations between the fatty acids were also
analyzed. One of them was found to be outstandingly
high, between C12:1 3OH and ECL12.35 (Pearson
coecient 0.97 when using percentage values, and 0.99
when using peak areas as variables), suggesting one
single group of bacteria behind the values of these
acids. The hypothesis was tested by performing a regression on the model: C12:13OH = A + B 
ECL12.35, which gives the following estimates for the
240 populations:
C12:1 3OH = 0.3% + 2.4  ECL12.35 (corrected
R 2 = 0.95; 95% con®dence interval for A: 0.2±
0.3% and for B: 2.3±2.4).
When the same model was used on the 109 isolates
forming Ps4±C4 we obtained:
C12:1 3OH = 1.0% + 1.9  ECL12.35 (corrected
R 2 = 0.80; 95% con®dence interval for A: 0.2±
1.9% and for B: 1.7±2.1);
and for 20 isolates from Ps4±C4 incubated under the
same conditions as the populations (Table 5):
C12:1 3OH = 1.1% + 2.0  ECL12.35 (corrected
R 2 = 0.83; 95% con®dence interval for A: 0.4±
1.8% and for B: 1.5±2.4).
To con®rm whether these results could possibly be

Table 3
The fatty acid composition in two distinct clusters of bacteria within
Group 1, Ps1±C1 (343 isolates) and Ps4±C4 (109 isolates)a
Fatty acids

C15:0 ISO 2OH&C16:1
C16:0
C18:1
C17:0 CYCLO
C12:0
C12:0 2OH
ECL12.35
C10:0 3OH
C12:0 3OH
C12:1 3OH

Ps1-C1

Ps4±C4

Group 2

Mean

SD

Mean

SD

Mean

SD

31.0
31.9
13.9
6.4
2.7
4.5
0.0
3.4
4.0
0.1

4.5
1.8
2.7
3.6
0.9
0.7
0.1
0.4
0.3
0.2

23.2
22.0
9.4
3.6
2.3
4.0
4.5
8.3
8.1
9.8

5.0
2.3
1.9
2.3
1.4
0.6
1.2
1.7
1.3
2.6

11.2
2.6
0.0
0.0
0.0
0.0
ndb
0.0
0.1
nd

6.5
1.9
0.2
0.1
0.5
0.1
0.1
0.4

a
Last column gives data for bacteria within Group 2. See text for
details.
b
Not detected.

Table 4
The relative content of fatty acids in 120 microbial populations originating from roots and rhizo-soil of barley plants sampled from plots
with crop rotation (CR) and barley monoculture (MC)a
Fatty acids

CR

MC

P-value

C15:0 ISO 2OH&C16:1
C16:0
C18:1
C17:0 CYCLO
C12:0
C12:0 2OH
ECL12.35
C10:0 3OH
C12:0 3OH
C12:1 3OH

30.9
13.7
15.6
4.7
2.6
2.5
0.4
2.8
2.8
1.3

31.6
14.7
15.8
4.8
2.6
3.2
0.8
3.3
3.1
2.1

nsb
0.003
ns
ns
ns
< 0.001
< 0.001
< 0.001
< 0.001
< 0.001

a
b

P-values from Kruskal±Wallis tests.
Statistically not signi®cant.

1449

S. Olsson, S. AlstroÈm / Soil Biology & Biochemistry 32 (2000) 1443±1451

statistical artifacts, the calculations were re-run with
the original values (peak area) instead of percentage
values. This test reveals a still closer correlation
between these two acids (corrected R 2 from 0.97 to
0.94 and B-estimates between 1.7 and 2.5)
The good agreement between the A- and B-estimates
for populations on one hand and isolates on the other,
corroborates the suggestion that the bacterial cluster
Ps4±C4 can explain the variation of C12:1 3OH and
ECL12.35 among the bacterial populations. When the
same model, however, is used to test the linkage
between C12:1 3OH on the one hand and C10:0 3OH
and C12:0 3OH on the other, no obvious correlations
were found among the 240 populations (corrected R 2
= 0.27 and 0.09, respectively). Thus, the high values
of these acids in MC soil probably have other sources
in addition to Ps4±C4.
The amount of C12:1 3OH was 2.0% in populations
from bulk soil as compared to 1.4% in those from
rhizo-soil (P < 0.001) sampled on the ®rst occasion.
On the second sampling occasion, the value 1.4% for
rhizo-soil had increased to 1.9% whether the inoculum
was diluted or not (P < 0.001). There was also a signi®cant interaction between experimental ®elds and rotational treatments with regard to proportion of C12:1
3OH; the MC e€ect being less pronounced in SaÈby
soil.
The higher contents of C16:0 and C12:0 2OH in
MC soil (Table 4) indicated a general lower frequency
of bacteria from Group 2 in these populations
(Table 3). This result, however, is mainly due to the
deviating low content of these acids in populations
from the CR plots at Ultuna (12% for C16:0 compared to 14±15% in all other plots) and a correspondingly higher content of C15:0 ISO and C15:0
ANTEISO which comprised 13% in CR soil from
Ultuna compared to 5±7% in all other plots (P <
0.001 under the hypothesis of no interaction between
experimental ®eld and rotational treatment). No such
interaction was found for C12:1 3OH, ECL12.35,
C10:0 3OH or C12:0 3OH. (The rotational e€ect on
C12:0 3OH should, however, be interpreted with some
caution as this fatty acid was a€ected by dilution of
the inoculum).
3.3. Some characteristics of bacteria in cluster Ps4±C4
All isolates in Ps4-C4 formed slimy colonies on TSA
and some of them produced a green pigment. The various tests on 22 di€erent isolates from this cluster
showed that they were ¯uorescent Gram negatives,
with an ability to metabolize cellulose. The tests for
proteolytic activity and phenol oxidation were negative. Of all the isolates tested, only four produced
HCN and most (90%) did not induce HR in the test
plant. About 80% were shown to be inhibitory to B.

sorokiniana (fungal growth after 15 days about 43 mm
compared to 65 mm in control) while none a€ected the
growth of R. solani (data not shown).
Detailed FAME pro®les of 20 isolates representative
of this cluster and conducted at two di€erent types of
incubation conditions are shown in Table 5. None of
the existing bacterial isolates could be identi®ed from
these pro®les.

4. Discussion
In the ®rst part of this study, it was possible to
recognize a group of bacterial isolates, Ps4±C4, present
with high frequency in barley MC soil and characterized by an unusually high content of the fatty acids
C12:1 3OH, ECL12.35, C10:0 3OH and C12:0 3OH.
In the second part, in which the fatty acid pro®les of
the culturable bacterial populations were analyzed, it
was further demonstrated that the MC populations
contained higher relative contents of these fatty acids
than the CR populations. A more detailed statistical
analysis of the pro®les of the populations revealed that
high prevalence of Ps4±C4 was sucient to explain the
high proportions of C12:1 3OH and ECL12.35 in MC
soil, and that bacterial groups other than Ps4±C4 had
to be assumed to explain the variations in C10:0 3OH
and C12:0 3OH.
The cluster Ps4±C4 was de®ned only by some
characteristics in the fatty acid pro®les and this does
not necessarily imply that all bacteria in it belong to
the same bacterial species. None of the isolates in Ps4±
C4 could be identi®ed with certainty by MIS although
Table 5
The fatty acid composition of 20 bacterial isolates typical of Ps4±C4
analyzed at two di€erent incubation conditions
Fatty acids

C15:0 ISO 2OH&C16:1
C16:0
C18:1
C17:0 CYCLO
C12:0
C12:0 2OH
ECL12.35
C10:0 3OH
C12:0 3OH
C12:1 3OH
ECL10.47
C10:0
ECL12.49
ECL15.26
ECL13.14
ECL12.52
a

Not detected.

5 g TSB and 88C

30 g TSB and 288C

Mean

SD

Mean

SD

32.9
18.4
19.3
7.2
4.1
3.7
1.4
4.6
4.4
3.8
0.0
0.0
nda
nda
nda
0.0

2.8
1.1
1.4
1.6
0.6
0.6
0.9
0.5
0.4
2.0
0.1
0.0

20.9
18.4
13.5
3.7
2.9
3.3
4.3
7.0
7.3
9.6
1.5
1.3
1.2
1.2
1.1
1.0

4.2
3.8
2.8
1.2
0.3
0.4
1.3
1.2
0.9
3.1
0.5
0.4
0.4
0.4
0.5
0.7

0.1

1450

S. Olsson, S. AlstroÈm / Soil Biology & Biochemistry 32 (2000) 1443±1451

some of them resemble Pseudomonas (SI 0 0.5). They
were all ¯uorescent but appeared morphologically
di€erent. Their ability to exude hydrolytic enzymes
such as cellulases is indicative of their ability to
actively penetrate plant tissues. At the same time,
absence of induction of HR by most isolates shows
that the plant recognizes them as non-pathogens.
Brie¯y, majority of the tested isolates (80±90%)
seemed to share a common pattern of functional
characteristics, which is clearly indicative of their common role in terms of ecological signi®cance in plant
rhizospheres viz. by providing protective shelter and
nutrient supplies by not killing their host. However,
more knowledge of their signi®cance and their identity
is needed to understand their unique association with
plants.
Stead (1992) investigated 340 isolates of plant pathogenic as well as other Pseudomonas species and found
that the proportion of 2- and 3-hydroxy fatty acids,
were the most useful parameters for classi®cation of
bacteria. According to data presented by Stead (1992),
the maximum value for C12:1 3OH, is 1.9% with its
mean at 0.2 for the group containing the highest content of hydroxy fatty acids. The corresponding mean
value in our studies was 9.4% (SD 4.1%) for Ps4±C4.
Furthermore, the sum of the three fatty acids C12:1
3OH, C12:0 3OH and C10:0 3OH in the same group
containing the highest content of hydroxy fatty acids
reported by Stead (1992) amounts to the mean value
8.1% as compared to 23.3% (SD 6.7%) for Ps4±C4 in
our study. This further indicates that the identity of
Ps4±C4 isolates is di€erent from that of other subgroups.
Cavigelli et al. (1995) analyzed FAME pro®les from
162 soil samples obtained from a corn ®eld in Michigan in USA. They analyzed in situ populations by
extracting fatty acids directly from the soil as well as
from cultivable populations by plating soil suspensions
on R2A agar. In this way, they registered a total of 56
di€erent fatty acids, none of which were C12:1 3OH.
Similarly Zelles et al. (1994) was not able to ®nd this
fatty acid when extracting directly from agricultural
soils in southern Germany. In comparison, an earlier
study (Olsson and Persson, 1999), based on 325 cultivable populations from seven di€erent ®elds of the
southern and central part of Sweden, showed that
most of these (320) contained C12:1 3OH (mean 1.9%;
SD 1.3%). (From basic data analyzed and partly presented in Table 3 in Olsson and Persson, 1999)
The discrepancy between our results and those of
Cavigelli et al. (1995) and Zelles et al. (1994) can not
possibly be explained by di€erences in the methods
chosen by each investigator. The main merit of extracting lipids directly from soil, i.e., the possibility to
detecting microorganisms which are dicult to culture
on nutrient agar, becomes a disadvantage of the

method based on extracting fatty acids from cultivable
populations due to the risk of losing information on
non-cultivable populations. Thus bacteria in Ps4±C4,
with its characteristically high content of C12:1 3OH,
ought to be detected by these investigators in extracting directly from soil. This conclusion ®nds support in
a statement made by Cavigelli et al. (1995) that ``the
distribution of a portion of the soil community that is
cultivable seems to be retained when soil communities
are cultured''.
One can argue that Ps4±C4 was selectively favored
by the incubation conditions used in our studies. In
fact, our data indicated that as much as 50% of the
lipids extracted from the cultivable populations originated from Ps4±C4, but only 10% of the isolates
belonged to this group. This reveals a serious drawback with analyzing cultivable populations, namely
that any incubation condition is bound to change the
in situ composition of the harvested biomass of bacteria. Nevertheless, it should have been possible to
detect the presence of C12:1 3OH even if Ps4±C4 represented only a small percentage of the bacterial in situ
biomass.
A more likely explanation for our results regarding
the frequency of occurrence of C12:1 3OH and Ps4±
C4 in agricultural soils of Sweden can be based on
di€erences in soil temperatures in Sweden compared to
elsewhere. As bacteria can adapt to lower temperatures
by forming a higher proportion of unsaturated fatty
acids (Rose, 1989), it is possible that Ps4±C4 contains
bacteria which are not usually found in soils of warmer areas. Whether this explanation is true can only
be proved in complementary investigations which systematically compare the in vitro and in situ fatty acid
pro®les of di€erent soils.
The fatty acid behind the ECL12.35 peak remains
unidenti®ed. There are, however, some indications that
it might be C11:1 3OH. C12:0 2OH, C12:1 3OH and
C12:0 3OH have, the equivalent chain length
(measured as retention time) of 13.18, 13.29 and 13.45,
respectively, in our equipment. The hydroxy acid
C11:0 2OH has 12.16 and C11:0 3OH has 12.44 and
ECL12.35 is in between them with 12.35. Similarly,
C12:1 3OH lies in between C12:0 2OH and C12:0
3OH. If this interpretation is correct, it can explain the
high correlation between C12:1 3OH and ECL12.35
for both individual isolates and microbial populations
and it suggests a metabolic linkage between the two
unsaturated 3-hydroxy fatty acids.
Steinberger et al. (1999) concluded that the hydroxy
fatty acids are useful for distinguishing between bacterial populations from di€erent soils and agricultural
practices, although their role in identi®cation of microorganisms in a population has not yet been described.
Our results con®rm both their conclusions, especially
with regard to cluster Ps4±C4. More, our analyses il-

S. Olsson, S. AlstroÈm / Soil Biology & Biochemistry 32 (2000) 1443±1451

lustrate the inherent possibilities in the approach of
combining fatty acid pro®les of individual bacterial
isolates with those of cultivated microbial populations
to draw valid conclusions.

Acknowledgements
We thank Paula Persson, Ann Gidlund and Lena
FaÈreby for all their advice and assistance with FAME
analyses. This work has been supported by research
grant from Federation of Swedish Farmers (SLF),
Stockholm.

References
AlstroÈm, S., Burns, R.G., 1989. Cyanide production of rhizobacteria
as a possible mechanism of plant growth inhibition. Biology &
Fertility of Soils 7, 232±238.
BoÈrjesson, G., Sundh, I., Tunlid, A., Svensson, B.H., 1998. Methane
oxidation in land®ll cover soils, as revealed by potential oxidation
measurements and phospholipid fatty acid analyses. Soil Biology
& Biochemistry 30 (10), 1423±1433.
Cavigelli, M.A., Robertson, G.P., Klug, M.J., 1995. Fatty acid
methyl ester (FAME) pro®les as measures of soil microbial community structure. Plant and soil 170, 99±113.
Curl, E., Truelove, B., 1986. The Rhizosphere. Springer-Verlag,
Berlin, p. 288.
Grayston, S.J., Wang, S., Campbell, C.D., Edwards, A.C., 1998.
Selecting in¯uence of plant species on microbial diversity in the
rhizosphere. Soil Biology & Biochemistry 30 (3), 369±378.
King, E.O., Ward, M.K., Raney, D.E., 1954. Two simple media for
the demonstration of pyocyanin and ¯uorescin. Journal of Lab.
Clinical Medicine 44, 301±307.
Klement, Z., 1963. Method for the rapid detection of the pathogenicity of phytopathogenic Pseudomonas. Nature 199, 299±300.
Kloepper, J.W., R-Ka'bana, R., McInroy, J.A., Collins, D.J., 1991.

1451

Analysis of populations and physiological characteristization of
microorganisms in rhizospheres of plants with antagonistic properties to phytopathogenic nematodes. Plant & Soil 136, 95±102.
Olsson, S., 1995. On barley monoculture soil: plant growth a€ecting
microbiota in soil from three long-term ®eld experiments on crop
rotation. Dissertation Swedish University of Agricultural
Sciences, Uppsala, Sweden.
Olsson, S., AlstroÈm, S., Persson, P., 1999. Barley rhizobacterial
population characterised by fatty acid pro®ling. Applied Soil
Ecology 12, 197±204.
Olsson, S., Persson, P., 1999. The composition of bacterial populations in soil fractions di€ering in their degree of adherence to
barley roots. Applied Soil Ecology 12, 205±215.
Rose, A.H., 1989. In¯uence of the environment on microbial lipid
composition. In: Ratledge, C., Wilkinson, S.G. (Eds.), Microbial
Lipids, vol. 1. Academic Press, London, pp. 255±278.
Sasser, M., 1990. Identi®cation of bacteria through fatty acid analysis. In: Clement, Z., Rudolph, K., Sands, D.C. (Eds.), Methods in
Phytobacteriology. AkadeÂmiai KiadoÂ, Budapest, pp. 199±204.
Stead, D.E., 1992. Grouping of plant-pathogenic and some other
Pseudomonas spp. by using cellular acid pro®les. Journal of
Systematic Bacteriology 42 (2), 281±295.
Steinberger, Y., Zelles, L., Bai, Q.Y., von, LuÈtzow, M., Munch, J.C.,
1999. Phospholipid fatty acid pro®les as indicators for the microbial community structure in soils along a climatic transect in
the Judean Desert. Biology & Fertility of Soils 28, 292±300.
Zelles, L., 1997. Phospholipid fatty acid pro®les in selected members
of soil microbial communities. Chemosphere 35 (1/2), 275±294.
Zelles, L., Bai, Q.Y., Beck, T., Beese, F., 1992. Signature fatty acids
in phospholipids and lipopolysaccharides as indicators of microbial biomass and community structure in agricultural soils.
Soil Biology & Biochemistry 24 (4), 317±323.
Zelles, L., Bai, Q.Y., Ma, R.X., Rackwitz, R., Winter, K., Beese, F.,
1994. Microbial biomass, metabolic activity and nutritional status
determined from fatty acid patterns and poly-hydroxybutyrate in
agricultural-managed soils. Soil Biology & Biochemistry 26 (4),
439±446.
Zelles, L., Rackwitz, R., Bai, Q.Y., Beck, T., Beese, F., 1995.
Discrimination of microbial diversity by fatty acid pro®les of
phospholipids and lipopolysaccharides in di€erently cultivated
soil. Plant & Soil 170, 115±122.