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.72000 Elsevier Science Ltd. All rights reserved.
Keywords:Rhizosphere bacteria; Barley; Monoculture; Hydroxy fatty acids
1. Introduction
The interface between soil and plant roots, the rhi-zosphere, 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 multipli-cation of a large variety of microorganisms. Dierent plant species release dierent organic compounds, and thus they can encourage a dierentiated rate of pro-liferation of the microbiota (Curl and Truelove, 1986;
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 diculties faced by many researchers while carrying out such investi-gations.
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 oers possi-bility of making a detailed description of the mem-bers of the communities. At the same time, a major
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drawback is that huge numbers of isolates are required to characterize and compare compositions of dierent communities. Similarly, an important advantage of working at the community level is that it may be easier to detect dierences between communities but it can then be dicult 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 dierences between the composition of bacterial populations in bulk soil and in the rhizo-sphere (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 bac-terial 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 diculty 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 diers from its ``in situ pro®le'' in soil. Therefore, it should be an advantage to analyze cultured commu-nities 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 prin-cipal aim was to ®nd and interpret dierences between rhizosphere microbial populations as an eect of long-term management practices due to dierent crop ro-tations. More speci®cally, we compared long-term bar-ley monoculture (MC) with a more diverse crop rotation (CR). Attempts were also made to study cer-tain phenotypic characteristics of the bacterial popu-lations speci®c to monoculture soils.
2. Materials and methods
2.1. Experimental ®elds
Soil or plant roots were sampled from three exper-imental ®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 ro-tation 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 oc-casions, 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, contain-ing 15 g lÿ1TSB (DIFCO) and 15 g lÿ1Oxoid
techni-cal agar). After incubating for 4±5 days at 118C, the bacterial isolates were sampled, approximately 15 iso-lates 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 occasions3 ®elds2 crop
ro-tations2 replicated plots x 3 replicated samples per
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 over-night. The suspensions thus obtained were spread on TSA5 (0.4 ml per plate, 5 g lÿ1TSB (DIFCO) and 15
g lÿ1 Oxoid technical agar) aseptically and left for
in-cubation 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 com-parison 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 eect 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 ana-lyzed from 120 soil-root samples collected on two oc-casions (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 incu-bated for 24 h at 248C. In contrast, the bacterial popu-lations were cultured on TSA5 before incubation for 5 days at 88C. These modi®cations were considered
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 popu-lations, 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 m0.2 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 com-ponent 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 pur-pose of comparison in this study, these peaks were given provisional names by referring to their equival-ent 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 thus-de®ned total named FAME peak area.
2.5. Bacterial tests
To determine a possible common pattern in func-tional 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 ¯uor-escence under UV light.
Isolates were further tested with respect to several phenotypes. Their ability to produce proteolytic enzymes was qualitatively analyzed by growing bac-teria 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 cel-lulolytic activity was done on cellulase indicator agar plates. This was done by culturing bacteria on TSA20 supplemented with Na-carboxymethylcellulose (5 g lÿ1
halo zones indicated production of cellulolytic enzymes.
In addition, phenol oxidation was tested by cultur-ing bacteria on gallic acid agar as described in Kloep-per 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 inter-preted by their ability to induce hypersensitive reaction (HR; Klement, 1963). HR was studied on freshly cul-tured bacteria suspended in sterile 0.1 M MgSO4 to
obtain a concentration of approx. 109 viable cells mlÿ1
after 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 cya-nide (HCN), was measured qualitatively as described in AlstroÈm and Burns (1989). Change in colour of in-dicator paper from yellow to brown revealed the pre-sence of HCN.
The biological activity of isolates was evaluated in terms of their ability to inhibit growth of fungal patho-gens. For this purpose, two pathogens, Rhizoctonia
solani KuÈhn and Bipolaris sorokiniana (Sacc.)
Shoe-maker 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 inocu-lated in the center of a PDA plate and bacteria were inoculated equidistantly from the fungus. After incu-bation 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 through-out. 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 dierences between iso-lates 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 dierence between bacteria from MC and CR soil. If the tests in step 2 were positive, i.e., if the
dierences 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 Cytophagaand Gram positives in Group 2.
3. Results
In the present study, the two bacterial groups, Group 1 and Group 2, were further analyzed separ-ately. Analysis of Group 1, which consisted of 720 iso-lates (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 dierentiated 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 iso-lates.
Isolates of Group 2 were also analyzed in the above manner but no indication of eect of rotational treat-ments on the FAME pro®les in this group was found (data not shown).
3.1. Bacterial groups
The composition of 720 isolates in Group 1 was further analyzed by dispersing the group into smaller clusters as follows:
Firstly the isolates were plotted over the ®rst two principal components (Fig. 1). The plots indicated that
the isolates could roughly be separated into ®ve sub-groups de®ned by:
Ps1: 0 < 1st component < 1 and 2nd component < 2
Ps2: 1st component > 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 corrobo-rated in a cluster analysis over the ®rst ®ve principal components (Wards linkage method of minimum
var-Table 1
The loadings of the dominating fatty acids on the ®rst and second principal components (PC)a
Fatty acids Loadings on the principal components Fatty acid content (%) P-value
1st PC 2nd PC CR MC
C15:0 ISO 2OH&C16:1 0.43 0.59 31.1 29.2 0.003
C16:0 0.58 ÿ0.44 29.2 28.8 nsb
C18:1 0.12 ÿ0.33 12.2 12.0 ns
C17:0 CYCLO 0.14 ÿ0.73 4.9 6.1 0.002
C12:0 0.31 0.81 3.6 3.3 ns
C12:0 2OH ÿ0.33 ÿ0.67 3.4 3.6 ns
ECL12.35 ÿ0.96 0.12 0.4 1.1 < 0.001
C10:0 3OH ÿ0.87 0.05 3.4 4.2 0.001
C12:0 3OH ÿ0.89 0.06 3.9 4.5 ns
C12:1 3OH ÿ0.97 0.12 0.9 2.3 < 0.001
a
The mean fatty acid compositions of bacteria originating from crop rotation (CR) and barley monoculture soil (MC) are also shown.P -values from Kruskal±Wallis tests.
b
Statistically not signi®cant.
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, respect-ively, 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 simi-larity 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 mi-crobial 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 evalu-ated by analyses of bacterial populations, which were obtained from roots of barley plants sampled directly from the experimental ®elds. The results are summar-ized 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 popu-lations.
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 coecient 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 re-gression 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
R2 = 0.95; 95% con®dence interval for A: 0.2± 0.3% and forB: 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
R2 = 0.80; 95% con®dence interval for A: 0.2± 1.9% and forB: 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
R2 = 0.83; 95% con®dence interval for A: 0.4± 1.8% and forB: 1.5±2.4).
To con®rm whether these results could possibly be
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 mini-mum variance method and Euclidean distances)
Ps1 Ps2 Ps3 Ps4 Ps5
C1 343 17 28 0 28
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 Ps1-C1 Ps4±C4 Group 2
Mean SD Mean SD Mean SD
C15:0 ISO 2OH&C16:1 31.0 4.5 23.2 5.0 11.2 6.5
C16:0 31.9 1.8 22.0 2.3 2.6 1.9
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 orig-inating 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 30.9 31.6 nsb
C16:0 13.7 14.7 0.003
C18:1 15.6 15.8 ns
C17:0 CYCLO 4.7 4.8 ns
C12:0 2.6 2.6 ns
C12:0 2OH 2.5 3.2 < 0.001
ECL12.35 0.4 0.8 < 0.001
C10:0 3OH 2.8 3.3 < 0.001
C12:0 3OH 2.8 3.1 < 0.001
C12:1 3OH 1.3 2.1 < 0.001
a
P-values from Kruskal±Wallis tests.
b
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 R2 from 0.97 to 0.94 andB-estimates between 1.7 and 2.5)
The good agreement between theA- andB-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 R2
= 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 sig-ni®cant interaction between experimental ®elds and ro-tational treatments with regard to proportion of C12:1 3OH; the MC eect 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 com-pared to 14±15% in all other plots) and a correspond-ingly 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 eect on C12:0 3OH should, however, be interpreted with some caution as this fatty acid was aected 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 var-ious tests on 22 dierent 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 nega-tive. 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 aected the growth ofR. solani(data not shown).
Detailed FAME pro®les of 20 isolates representative of this cluster and conducted at two dierent 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 character-ized 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 sucient 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 dierent incubation conditions
Fatty acids 5 g TSB and 88C 30 g TSB and 288C
Mean SD Mean SD
C15:0 ISO 2OH&C16:1 32.9 2.8 20.9 4.2
C16:0 18.4 1.1 18.4 3.8
C18:1 19.3 1.4 13.5 2.8
C17:0 CYCLO 7.2 1.6 3.7 1.2
C12:0 4.1 0.6 2.9 0.3
C12:0 2OH 3.7 0.6 3.3 0.4
ECL12.35 1.4 0.9 4.3 1.3
C10:0 3OH 4.6 0.5 7.0 1.2
C12:0 3OH 4.4 0.4 7.3 0.9
C12:1 3OH 3.8 2.0 9.6 3.1
ECL10.47 0.0 0.1 1.5 0.5
C10:0 0.0 0.0 1.3 0.4
ECL12.49 nda 1.2 0.4
ECL15.26 nda 1.2 0.4
ECL13.14 nda 1.1 0.5
ECL12.52 0.0 0.1 1.0 0.7
a
some of them resemble Pseudomonas (SI00.5). They
were all ¯uorescent but appeared morphologically dierent. 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 com-mon 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 patho-genic 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 con-tent 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 dierent from that of other sub-groups.
Cavigelli et al. (1995) analyzed FAME pro®les from 162 soil samples obtained from a corn ®eld in Michi-gan 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 dierent 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 culti-vable populations from seven dierent ®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 pre-sented 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 dierences in the methods chosen by each investigator. The main merit of extract-ing lipids directly from soil, i.e., the possibility to detecting microorganisms which are dicult 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 extract-ing 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 origi-nated from Ps4±C4, but only 10% of the isolates belonged to this group. This reveals a serious draw-back with analyzing cultivable populations, namely that any incubation condition is bound to change the in situ composition of the harvested biomass of bac-teria. Nevertheless, it should have been possible to detect the presence of C12:1 3OH even if Ps4±C4 rep-resented 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 dierences 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 war-mer areas. Whether this explanation is true can only be proved in complementary investigations which sys-tematically compare the in vitro and in situ fatty acid pro®les of dierent 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.
il-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.
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