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Keywords:

  • Endophytic bacteria;
  • Brassica napus;
  • Microbial diversity;
  • Cultivar difference;
  • Rahnella spp.

Abstract

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

Oilseed rape (Brassica napus) is one of the major oilseed crops in the world but is vulnerable to attack by many pathogens and insect pests. In addition to the host plant genotype, micro-organisms present in the rhizosphere and within plant tissues affect the susceptibility to plant pathogens. While rapid progress has been achieved concerning the concept of plant resistance genes, information on the role of the microbial community in plant protection is less apparent. We have studied the endophytic bacterial populations present in different tissues of oilseed rape and also analysed several cultivars (Express, Libraska, Maluka and Westar), which differ in their susceptibility to the wilt pathogen Verticillium longisporum. The population diversity was studied using agar plating assay, fatty acid methyl ester analysis and functional characterisation of isolated strains. Our work shows that already in the seeds there exists diversity in populations as well as in the total microbial load between two of the four tested cultivars. About 50% of the strains isolated from cultivars Express and Libraska showed moderate to strong direct inhibition of V. longisporum. The diversity of the endophytic flora isolated from oilseed rape and its implications in crop protection are discussed.


1Introduction

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

During recent years the important role of the soil micro-flora for proper and efficient development of crop plants has emerged. The micro-organisms in the soil can support plants in several ways such as assisting in supply of nutrients to the plants and inhibition of growth of pathogens. The beneficial effect of many soil bacteria has been experimentally verified and such bacteria are usually referred to as plant growth-promoting rhizobacteria (PGPR) [1–3]. It is also known that many fungi and bacteria colonise plant tissues internally and these are called endophytes provided that they do not cause any apparent harm to the host plant, as defined by Kado [4]. Endophytic bacteria that are found in the plant tissues are usually also present in the rhizosphere [5] as shown by Germida et al. [6] in oilseed rape and wheat. This suggests that migration of bacteria from the soil to the plant occurs via the roots.

It is generally accepted that all factors, e.g. crop rotation and soil amendments, that influence pathogen populations also have an impact on endophytic population structures [7]. Endophytic bacteria obtained from one host can also colonise other plant species internally [8–12]. This together with a possible selection of the micro-flora by the plant itself indicates that both generalists and specialists exist among endophytes.

Endophytic micro-organisms occur in many, if not all, crop plants. The diversity among endophytic bacteria has been illustrated by several reports during recent years. Fisher et al. [13] found that the diversity among endophytes decreased from the rhizosphere to the aerial part of the Zea mays plant, indicating that there is a selection of endophytes by the plant. As many as 46 different bacterial species from 27 different plant species were reported by Mundt and Hinkle [14] while 50 different bacterial species and 46 unidentifiable species were found in cotton and sweet corn [15]. Diversity has been found at the cultivar level as well. Studies conducted on endophytic colonisation of 11 field-grown pea cultivars showed significant differences in the level of endophyte colonisation between cultivars at the mature stage [16]. It is also known that tomato cultivars differ in their endophytic flora [17]. Further, Siciliano and coworkers [18] have shown that endophytic populations are different in the roots of Brassica napus cultivars Excel, Parkland and a transgene of Excel, Quest. Thus, there seems to be evidence in favour of a relationship between plant genotype and colonisation of endophytes, which requires comparative investigations also in other plant species and cultivars if, for instance, an optimal performance of introduced beneficial bacteria is to be obtained [19,20].

It has been shown that certain endophytes may serve as potential biocontrol agents against vascular pathogens [7,20,21]. There are few reports available concerning the presence of beneficial micro-organisms in oilseed rape, B. napus ([22,23,28,30]). However, they showed that both bacteria and fungi with biocontrol potential were present. Several fungal pathogens can cause serious damage to Brassica crops [24]. Verticillium longisporum (formerly V. dahliae, see e.g. [25]) is a devastating vascular pathogen on rapeseed crops causing wilt disease [26]. No efficient control measure of Verticillium is available. Fungicides are sometimes used to lower the impact of V. longisporum but are often not efficient since this is a soil-borne pathogen. One potential control strategy would be to employ naturally occurring endophytic micro-organisms to prevent or attenuate the disease in rapeseed.

We have, therefore, attempted to characterise and compare the endophytic bacterial populations present in some cultivars of B. napus. The four cultivars selected for this study, Express, Libraska, Maluka and Westar, are relatively genetically distant from each other and are known to possess different sensitivities towards fungal pathogens. The endophytic bacteria were isolated from these cultivars and characterised by analysing their functional properties and fatty acid methyl ester (FAME) profiles. FAME was used to characterise both populations and individual strains [27]. Earlier studies showed that several endophytes could inhibit the wilt pathogen and may therefore have an active role in resistance to disease. Therefore, the rapeseed bacteria obtained in this study were also tested for inhibition of growth of V. longisporum as an additional functional property.

2Materials and methods

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

2.1Organisms and growth conditions

Four cultivars of B. napus were used in this study. Two of these, Maluka and Westar, are both spring cultivars that are genetically relatively distant while the other two, Express and Libraska, are winter rape cultivars. Express is tolerant towards the fungal pathogen, V. longisporum, whereas Libraska is declared to be susceptible but also genetically relatively distant from Express (Norddeutsche Pflanzenzucht, Hans-Georg Lembke KG, Holtsee, Germany). The seeds of these cultivars were stored at 20°C while being used. This difference in susceptibility was verified in our greenhouse experiments (data not shown). Maluka and Westar differ in their sensitivity to Leptosphaeria maculans but are both susceptible to V. longisporum.

Pathogen-infested soil was prepared by inoculation of a pathogenic isolate of V. longisporum according to Alström [28]. The fungal isolate used for inoculations was obtained from a collection of pathogenic fungi maintained at the Department of Ecology and Crop Protection Science, SLU, Uppsala, Sweden. The pathogenicity of the isolate was confirmed on test cultivar Casino of B. napus by inoculating roots of 4-week-old plants before initiation of the greenhouse experiment and observation of wilt symptoms. Inoculum was prepared by culturing the fungus on potato dextrose broth containing phytagel (2 g l−1) in 9-cm Petri dishes and blending in deionised tap water just before inoculation (two dishes per 150 ml) as described in Alström [28].

The cultivars were grown in a greenhouse peat soil (Hasselfors Garden AB, Sweden) and sand mix (5:1). Growth conditions, watering and fertilisation of all plants were the same as specified earlier [28]. Two sets of plants were prepared, one in the presence of the pathogen and the other free of the pathogen. The pathogen was added by uniform watering with 20 ml of inoculum per pot 2 weeks after sowing as above and also outlined earlier [28]. Differences in the level of the wilt disease symptoms in the four cultivars were confirmed by recording the leaves showing drooping and development of chlorotic patches followed by necrosis. Leaves showing diffuse symptoms were incubated in moist chamber at 20°C for 4–5 days to favour the fungal growth and then observed for typical conidia and microsclerotia under a light microscope.

2.2Isolation procedure of endophytic bacteria

Endophytic bacteria were isolated from pre-germinated seeds, as well as from stem and root tissue of 6-week-old oilseed rape plants to investigate the bacterial diversity in the different cultivars. This experiment was repeated at least twice with different seed lots but since the results indicated the same pattern, as regards differences between the cultivars, to exist already in their pre-germinated seeds, the subsequent experiments were repeated only with pre-germinated seeds. Seeds were surface-sterilised in 70% ethanol during 1 min followed by thorough rinsing in sterile distilled water (SDW) and a final rinse in a sterile phosphate buffer saline solution (PBS; 0.14 M NaCl, 0.003 M KCl and 0.01 M phosphate buffer, pH 7.4) in the first experiment. In the subsequent experiments, the ethanol step was replaced with 20 min of continuous washing in SDW to minimise the contamination from the surface micro-organisms. The purpose was also to avoid negative effects, if any, associated with the chemical treatment on bacteria superficially present in the seeds. After testing the sterility, seeds were then pre-germinated aseptically on sterile filter paper soaked with SDW for 4–5 days in darkness at 20°C. Triplicate samples of at least 60 apparently vital pre-germinated seeds were crushed in 10 ml PBS. Suspensions thus obtained were diluted serially in PBS and then plated in duplicate on diluted TSA10 (10 g l−1 Tryptic soy broth from Difco, 15 g l−1 Oxoid technical agar) to enable estimation of total counts. Fluorescence was detected on King's medium B agar [29].

For isolation of endophytic bacteria from stem and root tissue, the lower part of three stems (the zone between the cotyledon attachment and the soil surface) and the upper part of three roots (the region below seed depth) were weighed and thoroughly rinsed in SDW as described above. The tissues were thereafter crushed in PBS and the suspensions thus obtained were spread on TSA10 as described above. All the inoculated plates were incubated at 20°C for at least 48 h before the colony-forming units (cfu) were counted. All the values were then transformed to cfu seed−1 or cfu g−1. Since only bacterial colonies grew on the plates under these conditions, no addition of antibiotics to the culture media was considered necessary.

2.3Sampling and characterisation of rapeseed endophytic bacteria

To facilitate comparison, bacterial colonies representative for each cultivar were sampled in a uniform way. The same dilution level from incubated TSA10 plates that reflected the quantitative and qualitative composition typical for each cultivar in terms of visual properties, e.g. size, colour and shape of colonies, was used. Bacteria were thus collected from all samples, which yielded a total of 58 bacterial strains. All these strains were stored in 20% glycerol at −20°C until use. To find any possible common pattern with regard to functional characteristics of sampled colonies within a cultivar, the colonies were isolated, purified and then examined in various ways. The functional characterisation of hydrolytic activity was carried out in terms of their ability to produce proteases, cellulases and phosphatases in vitro. Their ability to inhibit V. longisporum was also studied using an in vitro antagonism assay according to the procedure described in Alström [30]. Briefly, each strain was co-inoculated with V. longisporum on potato dextrose agar (two plates per combination) and incubated for 7–10 days at 20°C before degree of inhibition, if any, was recorded.

Strains from all the four cultivars, obtained as above, were subjected to further characterisation on the basis of their FAME profiles (see below for the procedure).

We also made an attempt to obtain bacterial populations that reflected the composition of each TSA10 plate and each cultivar, again with respect to size, colour and colony shape. Seed-borne bacterial populations were thus selected all at one sampling occasion from the same dilution. The bacterial populations were obtained by harvesting the bacterial mass from the surface of the agar plate in a uniform way with a plastic loop so as to obtain 50 mg fresh weight of bacterial mass per plate for FAME analysis. In total 12 bacterial populations (four cultivars×three repeats) were thus obtained for analysis from pre-germinated seed samples of the tested cultivars. These populations were also stored in sterile 20% glycerol until analysed for FAME profiles (see below). Use of TSA10 and the incubation temperature of 15°C for this purpose were considered necessary to mimic natural field conditions.

2.4FAME profile analysis

For fatty acid analysis of the sampled bacterial strains, the bacteria were grown on TSA according to the microbial identification system (MIS, Microbial ID, US) standard procedure (30 g l−1 Tryptic soy broth from Difco, 15 g l−1 Oxoid technical agar and 28°C). Approximately 50 mg fresh weight of bacterial mass of each strain was harvested and the FAMEs were extracted as described [31]. FAMEs were separated on a Hewlett Packard 5890 Series II gas chromatograph using a 25 m×0.2 mm methyl silicone fused silica capillary column, with hydrogen as carrier gas. Individual FAME profiles were identified and quantified using the peak-naming table component of the MIS. The relative quantities of different FAMEs were expressed as percentages of the total named FAME peak area. Isolates were identified by FAME profile analysis based on a SIM index value.

FAMEs from the bacterial populations were also extracted and analysed according to Sasser [31]. In general, for the purpose of comparison in this study, the peaks that could not be identified by MIS software were given provisional names by referring to their equivalent chain length. 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 defined total named FAME peak area [32]. Data was statistically analysed using variance analysis (Systat view 5).

3Results

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

We were able to isolate bacteria from all samples analysed while no fungi were recorded. The amounts of endophytic bacteria in pre-germinated seeds of the cultivars Express, Maluka and Westar were found to be approximately the same, but significantly higher than for Libraska (Table 1). The amount of bacteria in stem tissue was investigated in Libraska and Express grown in the absence of V. longisporum. In this case Express again seemed to contain a higher number of bacteria than Libraska grown in pathogen-free soil. However, the bacterial load increased in Libraska stems when the plants were grown in the presence of V. longisporum (Table 2). In general, Express also showed a higher abundance of proteolytic and cellulolytic bacteria than Libraska.

Table 1.  Average number of bacteria recovered from B. napus seeds in the cultivars Libraska, Express, Maluka and Westar
  1. *Significantly different from others at P=0.05 (log-transformed).

B. napus cultivarNumber of bacteria g−1 seed (×107)Number of bacteria seed−1 (×105)Average weight of 1 seed (g)
Libraska2.5*1.1*0.0043
Maluka8.53.10.0036
Express9.44.2*0.0045
Westar6.92.80.0041
Table 2.  Bacterial populations isolated from seeds and stems of B. napus cultivars grown in the presence or absence of V. longisporum under greenhouse conditions
 Express (tolerant) ×105 cfu g−1Libraska (susceptible) ×105 cfu g−1Express:Libraska ratio
Soaked seeds (no soil)
Total21.81.7512.5
Proteolytic≫1.00.77≫1.3
Cellulolytic≫1.00.65≫1.5
Stem (Verticillium-free soil)
Total0.050.0095.6
Proteolytic0.0250.0151.7
Cellulolytic<0.0010.0350.03
Stem (Verticillium-infested soil)
Total0.918.50.11
Proteolytic0.760.0419
Cellulolytic0.430.0656.6

FAME analysis of the single strains from pre-germinated seeds revealed that the cultivars Maluka and Westar basically carried the same families of bacteria, i.e. the microbial diversity in these two cultivars was smaller than for Express and Libraska (Table 3). The results also showed that Express seeds carried predominantly Pseudomonas spp. and that Libraska seeds contained bacteria from the genus Bacillus in addition to Pseudomonas spp. The seed treatment procedure (sterile water v. 70% ethanol) had no apparent negative effect on cfu g−1 tissue recovered, but it was found to affect the composition of the endophytic bacteria isolated (Tables 3 and 4). When seeds were washed with 70% ethanol we recovered mainly Rahnella aquatilis strains from Express seeds (Table 4). On the other hand, R. aquatilis strains were found in only four out of 29 isolated strains when seeds were thoroughly washed with SDW (Table 3).

Table 3.  Endophytic bacteria isolated from pre-germinated seed from B. napus cultivars Express, Libraska, Westar and Maluka
  1. Seeds were washed in sterile water for 20 min prior to pre-germination and extraction (see Section 2).

Bacterial strainPre-germinated seeds from
 ExpressLibraskaWestarMaluka
Alcaligenes piechaudii1   
Bacillus amyloliquefaciens 1  
Bacillus brevis 1  
Bacillus megaterium 3  
Comamonas acidovorans 1  
Chryseobacterium indologenes    
Erwinia amylovora1   
Kurtia gibsonii 1  
Micrococcus luteus 4  
Pantoea agglomerans7   
Pseudomonas chlororaphis  32
Pseudomonas fluorescens21  
Pseudomonas marginalis331616
Pseudomonas putida1071517
Pseudomonas syringae1   
Rahnella aquatilis4   
Not identified18  
Total number of strains tested30303435
Table 4.  In vitro characteristics of endophytic bacteria isolated from different plant tissues of a wilt-tolerant (Express) and a wilt-susceptible cultivar (Libraska) of B. napus, grown in the presence or absence of the pathogen V. longisporum
  1. a−, +, ++, +++ indicate no, presence, moderate and strong activity, respectively.

  2. b− and + represent absence and presence of activity, respectively.

  3. cBased on FAME profile analysis.

SourceBacteriaSimilarity index valuecDirect antagonism (V. dahliae)aFluorescencebPhosphatase activitybProtease activitya
Libraska,1-Cytophaga johnsonae0.03+++
root,2-Aureobacterium barkeri0.61+
Inf. soil3-Cytophaga johnsonae0.26++
 4-Acidovorax Delafieldii0.97++
 5-Cytophaga johnsonae0.88+++++++
 6-Agrobacterium rubi0.89++++
 7-Pseudomonas fluorescens0.65++++
 8-Not identified ++++++++
Libraska,1-Not identified +++
stem,2-Not identified +
Inf. soil3-Not identified +
 4-Not identified +++
 5-Not identified +++
 6-Not identified +++
Libraska,1-Chryseobacterium balustinum0.79+++
stem,2-Paenibacillus gordonae0.54+++
healthy soil3-Chryseobacterium indologenes0.86+++
 4-Bacillus mycoides0.36++++
 5-Paenibacillus polymyxa0.50+++++
 6-P. polymyxa0.58+++++
 7-P. polymyxa0.65+++++
Libraska,1-P. putida0.70+++++
root,2-P. putida0.72+++++
healthy soil3-Chryseobacterium indologenes0.93++++
 4-P. aeruginosa0.72++
 5-P. putida0.92++
 6-P. putida0.96+
Libraska,1-Bacillus licheniformis0.74++++
seed2-B. mycoides0.24++++
 3-B. licheniformis0.72++++
 4-Acinetobacter calcoaceticus0.24++
 5-A. lwoffii0.80+
 6-B. mycoides0.23++++
 7-Rahnella aquatilis0.74++
Express,1-Not identified ++++++
root,2-Not identified +++
Inf. soil3-Not identified +++
 4-Not identified ++
 5-Not identified ++
 6-Not identified ++++
Express,1-Not identified ++
stem,2-Not identified +
Inf. soil3-Not identified ++
 4-Not identified +
 5-Not identified +++
 6-Not identified ++
Express,1-Not identified +++
stem,2-Not identified +++
Inf. soil3-Not identified +++
 4-Not identified +
 5-Not identified ++
 6-Not identified +++
Express,1-Rahnella aquatilis0.69++
seed2-R. aquatilis0.58++
 3-R. aquatilis0.79++
 4-R. aquatilis0.56++
 5-R. aquatilis0.80++
 6-R. aquatilis0.59++

Results from the FAME analysis of the bacterial populations in pre-germinated seeds divided the four cultivars into two groups. This grouping was mainly based on the presence of the fatty acid 17:0 CYCLO (provisional name F1689), but also on F1581 (provisional name for a group of three fatty acids that was inseparable) and 19:0 CYCLO W8C (omega, provisional name F1890). Express and Maluka grouped together while Libraska grouped with Westar (Fig. 1). Further, the bacterial population of Express seemed to differ slightly from the population of Maluka. FAME analysis performed on the individual strains further confirmed the separation of Express from Libraska based on the cyclic fatty acid 17:0 CYCLO. This fatty acid was found in both cultivars but usually in higher amounts in Express than in Libraska (data not shown). Besides, Libraska bacteria were found to contain higher levels of two odd fatty acids, 15:e ISO and 15:0 ANTEISO, than the bacteria isolated from Express.

image

Figure 1. FAME analysis on endophytic bacterial populations isolated from pre-germinated seeds of B. napus cultivars Express (E), Libraska (L), Westar (W) and Maluka (M). The provisional name F1689 represents the fatty acid 17:0 CYCLO while F1581 is the provisional name for a group of three fatty acids that were inseparable.

Download figure to PowerPoint

Bacterial strains from the two microbially different cultivars Libraska and Express were further characterised in different ways and the results are summarised in Table 4. The strongest in vitro inhibition of V. longisporum was demonstrated by Paenibacillus polymyxa (as identified by FAME) but no indirect inhibition could be seen. This bacterium was isolated from the stem of Libraska grown in healthy soil in the greenhouse and was found in three out of seven strains. Further, of the bacterial strains isolated from Express, 54% possessed a moderate to strong ability to inhibit V. longisporum compared to 48% for strains from Libraska. Express also contained a higher proportion of bacteria with protease activity, 58%, compared to the ratio of 17% in Libraska (Table 4).

4Discussion

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

In this study, we found noticeable differences in the endophytic bacterial populations recovered from the B. napus cultivars Express and Libraska, while the bacterial populations from the cultivars Maluka and Westar were more similar to each other. Libraska is known to be susceptible and Express tolerant to V. longisporum, whereas Maluka and Westar are equally sensitive towards this pathogen. The differences between Express and Libraska populations in pre-germinated seeds were also established both qualitatively and quantitatively. Studies carried out by Elvira-Recuenco and Vuurde [16] on endophytic colonisation of 11 field-grown pea cultivars showed significant differences in the level of endophyte colonisation between cultivars at the mature stage. Express cultivar contained a higher proportion of bacteria having functional characteristics with regard to proteolytic and cellulolytic activity besides phosphatase activity than Libraska. These characteristics among PGPR are related to their nutrient-acquiring ability and to some extent also to their plant protection ability [7].

Many of the isolated strains belonged to genera, e.g. Pseudomonas, Bacillus, Alcaligenes, that are already known as PGPRs. Their indirect beneficial effect on plants is partly attributed to induced systemic resistance, a mechanism among others explaining the biocontrol effect of many PGPRs [33]. Whether this mechanism is operating here remains to be established for the bacteria isolated from the studied cultivars. However, there are several examples of plant-protecting Bacillus and Pseudomonas that are not only beneficial for plants but also endophytic in nature [34]. The endophytic population from Libraska in our study contained more strains of Bacillus spp. compared with that from Express. Strains belonging to Pseudomonas were also identified from Libraska and it can be speculated that strains present in this cultivar may have a protection potential that can be utilised in other cultivars. We have indications from preliminary investigations that the endophytic populations from Express have a beneficial potential in terms of disease suppression and/or plant growth-promoting effect on Libraska, using population cross-inoculation experiments. How much these genera and other genera with properties of PGPRs found in the studied cultivars really contribute to disease protection is yet to be investigated.

No fungal colonisers were observed in the plant material used in this study. However, the technique used for isolation of bacteria in our study did not favour growth of fungi. Investigations done by Berg [23] on different cultivars, using rape root extract-based media, showed the presence of both fungi and bacteria from B. napus. We were able to detect at least 10 genera and 16 species of endophytic bacteria from pre-germinated seeds of the tested cultivars compared to other reports showing a broad diversity in the absence of soil. Siciliano et al. [18] recovered at least 25 genera, including Pseudomonas and Bacillus, from roots of B. napus and B. rapa indicating a high microbial diversity as expected in roots. Bacterial groups colonising B. napus in that study were also detected as inhabitants of the cultivars tested here. It is known that the same cultivar of peanut grown in different fields showed a significant difference in endophytic diversity in peanut plants [35]. We cannot exclude that the lower diversity in the endophytic populations recovered from B. napus cultivars Maluka and Westar may depend on prior conditions of propagation. The potential impact of different culturing conditions on the microbial diversity is currently being investigated using different cultivars.

We noticed a relatively good agreement between results obtained from FAME profiles of strains, in vitro studies on microbial diversity, and FAME of populations in the case of Libraska and Express. This correlation was not evident with the cultivars Maluka and Westar as we observed a difference in the FAME profiles of their entire populations compared to what was expressed in the in vitro agar assay. It is possible that culturable endophytic strains actually were present but in undetectably small numbers on the agar media used and thus prohibited detection and isolation. Accordingly, FAME analysis of bacterial populations is to be preferred to agar assay as it offers the possibility to efficiently explore cultivar differences with respect to microbial diversity.

Use of bacterial endophytes for potential biocontrol of fungal or bacterial diseases has been investigated for only a limited number of plant species. Being inside the plant, the endophytic bacteria are in a favourable situation to promote plant health. The same bacterium may cause both growth promotion of the host and biological control of pathogens. Evidence on biocontrol activity of endophytic bacteria against fungal pathogens using Pseudomonas and Bacillus comes from several studies on crops such as cotton, oilseed rape, potato, tomato, cucumber and pea [30,36–40]. Van Buren et al. [41] found 61 out of 192 endophytic bacterial strains recovered from potato stem tissue to be effective as biocontrol agents against Clavibacter michiganensis subsp. sepedonicus. One of their strains, a fluorescent pseudomonad CICA90, colonised roots as well as stems both internally and externally. Nearly all strains from Libraska and Express in this study were found to exude growth-inhibitory substances towards V. longisporum when tested in vitro, nevertheless the degree of inhibition was cultivar- as well as strain-dependent. It is possible that microbial metabolites may have an active role in resistance to disease by functioning as signals mediating a cross-talk between the endophyte and its host. One important factor required for an optimal performance of an introduced endophytic micro-organism is, however, considered to be the relationship between plant genotype and effective colonisation of the host [19,20].

A successful outcome of biological control in the field demands a better understanding of the complex microbial interactions in plants. Cultivar differences in terms of resistance to pathogens may partly be due to qualitative and quantitative differences in their endophytic microbial populations. Therefore the potential of endophytic strains naturally colonising plants should probably be given greater attention in plant breeding but calls for further studies of microbial ecology and pathology.

Acknowledgements

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

Svalöf Weibull AB, Sweden kindly provided oilseed rape seeds. Anna Keifer is thanked for timely technical assistance and Dr Stig Olsson for help with FAME analysis and statistics. This study was financially supported by the Swedish Research Council for Forestry and Agriculture, The Royal Swedish Academy of Agriculture and Forestry and Carl Tryggers Stiftelse, Stockholm.

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  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References
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