Competition between Pseudomonas fluorescens Ag1 and Alcaligenes eutrophus JMP134 (pJP4) during colonization of barley roots

ELSEVIER

FEMS

Microbiology

Ecology

20 (I 996) 4 l-5

I

Competition between Pseudomonas fluorescens Ag 1 and Alcaligenes eutrophus JMP134 ( pJP4) during colonization of barley roots Lene Kragelund * , Ole Nybroe Microbiology

Srctiorl.

Department

Received

of Emlog?

and Molecular DK-1958

1 November

Biology, The Royal Veterinoq Frederiksberg C, Denmark

1995: revised

6 February

1996; accepted

and Agricultural

12 February

lJnicersit);,

Ro[igh&LVj

21,

1996

Abstract To use deliberately released beneficial microorganisms in the rhizosphere, we need a better understanding of the process of root colonization by seed-borne or soil-borne inocula. In this study, we determine the survival of Pseudomonas Juorescens Agl and Alcaligenes eurrophus JMP134, their colonization ability as affected by substrates, and the relative importance of migration versus competition for colonization of the root. Agl and the 2,4-dichlorophenoxy-acetic acid (2,4-D) degrader JMPl34 were inoculated in sterile barley rhizosphere systems. After inoculation of seeds with individual strains, comparable population sizes were established in the rhizosphere as determined by immunofluorescence microscopic total cell counts. Both strains were motile and able to colonize the entire root system without percolating water to stimulate passive transport. Comparing immunofluorescence microscopic cell counts with colony-forming units demonstrated that a subpopulation of A. eutrophus JMP134 closely associated with the root was non-culturable in contrast to the population in rhizosphere soil. Hence, the sole use of culture-dependent methods may give misleading information about the distribution of bacteria in the rhizosphere. Colonization studies with both strains showed that co-inoculation of Agl and JMP134 caused a decrease of the population size of JMPl34 if 2,4-D was not added to the soil as a specific carbon source for this strain. Thus, competition for limited carbon sources might influence the composition of the bacterial community in the rhizosphere. We also found that the presence of an established inoculum in the soil reduced subsequent root colonization by a seed-inoculated strain, probably by filling available niches, also indicating that competition from other bacteria may be an important factor determining the distribution of seed-borne inocula. This factor may be just as important for the distribution of bacteria as migration. Keywords:

Root colonization;

Competition;

2,4-Dichlorophenoxy-acetic

1. Introduction Introduction of beneficial microorganisms into the rhizosphere environment may represent a way to * Corresponding 28 26 24; E-mail:

author. Tel: +45 35 28 26 30; Fax: [email protected]

0168-6496/96/$15.00 PII

SO168-6496(96)00013-X

0 1996 Federation

of European

+45

35

Microbiological

acid

improve agricultural and environmental quality of a habitat. For example, several root-colonizing fluorescent Pseudomonas strains are associated with increased plant growth [l-3] and the rhizosphere might also represent a favourable environment for bioremediation [4]. To exploit the beneficial microbial properties in the rhizosphere, we need a better underSocieties.

All rights

reserved

42

L. Kragelund,

0. Nyhroe

/ FEMS

standing of the process of root colonization by seedborne or soil-borne inocula [5-71. One of the important steps during colonization of the root by introduced microorganisms is the migration or transport from the point of application towards the roots and downwards during root extension. Percolating soil water and channels created in the soil by growing plant roots can provide passive transport [8-lo], whereas active migration is due to bacterial motility. Some results indicate that nonmotile mutants are poorer colonizers than the corresponding wild types [ 11,121 and that chemotaxis towards exudates might be important [13,14]. However, others have found that motility gives no advantage during colonization [ 10,151 so the significance of motility and chemotaxis to successful root colonization remains unclear. Adherence to the root tissue is considered to be important for rhizosphere colonization [5,16]. Information is, however, conflicting about the adhesion mechanisms. Some researchers have suggested that the rapid adherence of Pseudomonas to roots is due to nonspecific binding [5], whereas others have identified a specific adhesion mechanism in P. putidu [161. Primary root colonizers such as Pseudomonas strains are able to grow rapidly on simple sugars and carboxylic acids, compounds known to be abundant in exudates [ 171. Therefore they might be able to fill available physical and nutritional niches in the rhizosphere, hence impairing the establishment of other bacteria. However, herbicides, which are routinely applied in normal agricultural practice may provide a selective advantage to herbicide degraders and might alter the nutritional conditions in the rhizosphere, e.g. due to changes in root exudation [ 181. In studies of seed-borne inoculants it has frequently been found that colonization of the lower parts of the root is poor and may be favoured by soil inoculation [7, lo]. Any of the traits mentioned above might differentially influence soil- and seed-inoculated bacteria, but at the present time conclusions about the mechanisms involved cannot be drawn. In the present study we compared the colonization of root tip and root base segments of barley seedlings by two strains with different growth requirements: Pseudomonas jluorescens Ag 1 and Alcaligenes eutrophus JMP134. Agl is a denitrifying strain able to

Microbiology

Ecology

20 (19%)

41-51

exploit numerous low-molecular substrates, whereas JMP134 is a soil bacterium with a limited substrate spectrum but able to degrade 2,4-dichlorophenoxyacetic acid (2,4-D) that has been widely used in agriculture as a herbicide. Both strains have been model strains for several studies addressing the ecology of bacteria released into natural environments [ 19,201. In a series of competition experiments, we studied how (1) soil versus seed inoculation and (2) addition of 2,4-D as a selective substrate affected the distribution of the two strains along the root and their attachment to the root tissue. During these experiments we determined both the total cell numbers by immunofluorescence microscopy and the culturable cell numbers by colony blotting to obtain further information about cell viability in different rhizosphere habitats.

2. Materials

and methods

2. I. Bacteria and growth conditions Two strains of bacteria were used in this study: Pseudomonas jluorescens Ag 1 isolated from grassland soil by Dr. S. Christensen, Copenhagen University, Denmark and me 2,4-D degrading soil bacterium Alcaligenes eutrophus JMP134 (pJP4) [21]. Analysis by Biolog GN showed that P. jluorescens Agl is able to utilize 63 of the 95 carbon sources tested, whereas A. eutrophus JMP134 can only use 48. The most conspicuous difference was that Agl can utilize 11 sugars while JMP134 can utilize only fructose. At standard conditions the bacteria were grown aerobically in 25 ml of Luria Broth, pH 7.2, 0.4% glucose (LB: 1% tryptone, 0.5% yeast extract, 1% NaCl). The cultures were grown at room temperature (22°C) on a rotary shaker at 120 ‘pm. Both strains were motile. The two strains were not antagonistic to each other when tested on LB agar and the growth of P. jluorescens Agl was not impaired by including 10 ppm of 2,4-D in the growth medium. 2.2. Soil The soil used was a sandy loam (pH of soil water: 6.5) taken from a field at The Royal Veterinary and

L. Kmgelund,

0. Nybme

/ FEMS Microbiology

Agricultural University, Hojbakkegkd, T%strup, Denmark. The soil had been cropped to barley. The soil was stored in plastic bags at 15°C without drying. The soil was then sieved ( < 2 mm) before use and sterilized by autoclaving in boiling tubes for 60 min at 121°C on three consecutive days. Sterility of the soil was checked by plating subsamples of the autoclaved soil on LB agar and incubation for 7 days at 25°C. Soil moisture was adjusted to 15% (w/w) with sterile water. In some experiments, 2,4-D was added together with the water to 10 pg 2,4-D/g soil. 2.3. Seeds and surface sterilization Barley seeds (var. Digger) were surface-sterilized by immersion in 70% (v/v) ethanol for 1 min, followed by treatment in 3% (v/v> hypochlorite for 1.5 h under vacuum for the first 10 min. After being repeatedly rinsed in sterile water (2 h), the water was removed and the seeds were ready to use. Several seeds from each batch were selected for a sterility test. They were incubated on LB-agar at 25°C for several days without any contamination appearing. 2.4. Colonization assays The ability of the bacteria to colonize plant roots was determined by inoculation of surface-sterilized barley seeds. Surface-sterilized barley seeds were aseptically transferred to 10 ml bacterial suspension (late logarithmic phase) adjusted turbidimetrically to a density of 5 X lo9 cells ml-‘. The cells were washed twice with sterile 0.9% NaCl prior to use. When the seeds were inoculated for 30 min in the cell suspension containing 5 X lo9 bacteria ml-‘, for both strains about 10’ bacteria were found on each seed when the seeds were removed from the bacterial suspension without rinsing. The seeds were planted in sterile 50-ml plastic tubes that were filled with 50 g of sterile soil (15% water). Three tubes were placed in a beaker with 300 g soil (15% water) and the whole system was placed in a sterile 500-ml transparent plastic bag to prevent soil drying. The systems were incubated at 20°C (12 h dark/l 2 h light) for 7 days. No significant water loss from the tubes was seen during the 7-day period. A competition assay was made where seeds were

Ecology

20 (1996141-51

43

inoculated in a bacterial suspension containing an equal concentration (5 X 10’ cells ml- ’ > of both Agl and JMP134. Otherwise, the procedure was as described above. In another competition assay, both strains were inoculated in the soil at a concentration of lo* bacteria gg’. Seeds were planted after three days. In a third competition assay, the soil was inoculated with one of the strains at a concentration of 10’ bacteria gg ‘. After three days, the seed was coated with the other strain and planted in the pre-inoculated soil. The seed inoculation was as described above. Each of the above experiments included four to six plants. In an attempt to favour A. eutrophus JMP134 an experiment with four plants was made where 2,4-D was added to the soil as described above and both Agl and JMP134 were inoculated onto the seed. 2.5. Seedling harvest and recovery of bacteria Two methods to release bacteria from the roots were compared. These were mild sonication in an ultrasonic waterbath (Metason 200, Struers, Denmark) and agitation on a whirly-mixer. Both procedures were performed for 15 s, 40 s, and 2 min. The two methods gave comparable results, and extended sonication/mixing did not increase the recovery. None of the methods affected the culturability of any of the strains. Mild sonication for 15 s was used in the colonization experiments. The procedure for homogenizing root fragments was optimized as well. Three procedures were compared: (a) blending in an ultra turrax (Ultra Turrax T25, IKA-labortechnik, Germany), (b) homogenization in a morter, and (cl a combination of the two methods. In the colonization experiments, the roots were homogenized by the combination method, as this gave the highest recovery of cells. When the plants were harvested from the soil system, the roots were divided into segments: an upper 1 cm fragment adjoining the stem and a lower 1 cm fragment including the root tip. About 5 mg of soil adhered to each root fragment. Three root pieces were sequentially transferred under sterile conditions through 5 washes with 1 ml phosphate buffered saline (PBS) per centimetre of root. Afterwards, the roots were homogenized in 1 ml PBS per centimetre of root.

2.6. Immunojluorescence microscopy

Axioplan epifluorescence microscope at a 1250 X magnification.

Immunofluorescence microscopy was used to determine the total number of Agl or JMPI 34 cells. Samples for immunofluorescence microscopy (IFM) were fixed by adding formaldehyde to a total concentration of 2%. In brief, bacteria from the washing fractions or the homogenate were collected by filtration onto a 0.2 /*rn black polycarbonate filter (Nuclepore, USA). After three washes to remove cell debris, the filter was incubated 1 h with the strainspecific primary antibody (anti-Agl or anti-JMPl34). Anti-Agl recognized specific Agl [22] and antiJMP134 was strain-specific against JMP 134 [23]. After that, the unbound primary antibody was washed off and the filter was incubated with FITC-conjugated sheep anti-rabbit immunoglobulins diluted I:20 (Dako, Denmark) for 1 h. The excess of the secondary antibody was removed by washing, and the filter was placed on a microscope slide on a drop of No Fade medium (10 ml PBS, 90 ml glycerol, 100 mg paraphenylenediamine, 0.5 M carbonate/bicarbonate buffer, pH 8.0) [24]. A cover slip with a thin film of No Fade medium was then placed on the filter. Labelled cells were enumerated using a Zeiss Root base

2.7. Colony blotting The number of culturable Pseudomonas jluorescens Agl and Alcaligenes eutrophus JMP 134 were determined by duplicate plating of appropriate dilutions. Portions (0.1 ml) of the washes and the homogenate were diluted and spread plated onto LB with 1.5% agar. Colonies were counted after 2 days of incubation at 30°C. In competition experiments, colony blots of the plates were performed as described by Kandel et al. [23] using the antibodies mentioned above. 2.8. Statistical analysis All colonization experiments were carried out with 4-6 plants. Student’s t-test was used to test differences between two treatments using the statistical program SigmaStat. If either a normality test or an equal variance test failed, the Mann-Whitney Rank Sum test was used. A P value < 0.05 was consid-

Root tip

A

le+7 i

le+7 hll 1e+6

1 e+6

1 e+5

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le+l

le+O

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Washmg fractions

Fig. I. Seeds were inoculated with either P. jluorescms fractions 1. and 2.) and the rhizoplane (washing fractions by immunofluorescence microscopy and spread plating. errors.

m

lmmunofluorescence

0

Colony formlng units

mIcroscopy

horn

1. 2

Washing

fractions

5

horn

counts

Agl (A) or A. rurrophus JMPl34 (B). Bacteria from the rhiroaphere (washing 5. and the homogenate (horn)) were harvested and the cell number was determined Each data set represents the mean of results from six plants. Bars indicate standard

ered necessary to establish a statistically significant difference between treatments.

counts in all fractions at the root base, and at the root tip the culturability of JMP134 closely associated to the root (wash 5 and homogenate) was very low (Fig. lB>.

3. Results 3.1. Establishment of Pseudomonas jkorescens Agl and Alcaligenes eutrophus JMP134 in the sterile rhizosphere Sterile barley seeds were inoculated with P. ,fluorescen.s Agl or with A. eutrophus JMP134 and planted in sterile soil. After one week, the distributions of Agl and JMPI 34 on the developing root system were determined by immunofluorescence microscopy and by spread plating. When assayed by immunofluorescence microscopy, the first two washes of the root base segments contained I .5 X I O7 and I. I X IO6 Agl cells cm root-‘, and the corresponding cell counts for JMP134 were 1.2 X IO’ and 1.l X lo6 cells cm root-’ (Fig. 1). These washes were rich in soil particles and are hence considered to represent rhizosphere soil. The fifth wash was devoid of soil particles and contained 8.4 X 10’ Agl cells cm- ’ and 8.9 X 10” JMP134 cells cm-’ while 3.8 X IO5 Agl cells cm-’ and 1.7 X 10’ JMP134 cells cm-’ were found in the root homogenate. The latter two fractions probably contain cells that have been firmly attached to root tissue or have colonized the endorhizosphere. At the root tip (mean root length ca. 7 cm>, the population sizes were lower than at the root base (Fig. 1). Only 3.3 X lo6 and 2.5 X lo5 JMPI 34 cells were found per cm root in the first and second wash, respectively, 2.1 X 10” Agl cells cm-’ and 2.7 X 10’ JMP134 cells cm-’ in the fifth wash, and 2,4 X 10” Agl cells and 1.4 X 10” JMP134 cells per cm root in the homogenate of the root-tip. Hence both strains were able to establish themselves over the total root system and the distribution of the two strains appeared quite similar. Detection of P. jluorescens Agl by the spread plate method showed that it remained fully culturable during the experimental period of seven days. No significant differences in the amount of Agl detected by immunofluorescence microscopy or plate counting were observed neither at the root base nor at the root tip (Fig. 1A). Culturable counts of A. eutrophus JMP134 were slightly lower than total

3.2. Competition co-inoculation

between Agl

and JMP134 qfter

Fig. 1 showed that Agl and JMP134 established comparable total population sizes in barley rhizosphere when inoculated alone on the seed. In the next series of experiments the two strains were coinoculated onto the seed to determine any competitive interactions between them. Tables 1 and 2 (exp. A and B 1) show that co-inoculation on the seed affected neither the population size nor the distribution pattern of Agl compared to single inoculation. In addition, the number of culturable cells equalled the total cell number, even in this experiment (not shown). In contrast, JMP 134 established a slightly reduced population in the presence of Agl, Tables 1 and 2 (exp. F and B2). The inhibition of JMP134 was most pronounced at the root tip, where cell numbers in all fractions were reduced more than ten-fold. At the root tip, the culturability of JMP134 closely associated to the root was low as seen in the single inoculation experiment (not shown). Co-inoculation of both strains in the soil resulted in a distribution pattern resembling that obtained after co-inoculation on the seed. The Agl population was always higher than the JMP134 population, and for all fractions at the root base this difference was statistically significant (Tables 1 and 2, exp. Dl and D2). 3.3. Competition between Agl mixed seed-soil inoculation

and JMP134 gfrer

The next round of experiments were performed to assess the competition between the two strains when one strain was introduced into the soil and the second strain was inoculated on the seed that was planted three days later. As shown in Table 1 (exp. A, B 1, Cl), significantly less Agl were present in the first and second washes, when JMPI 34 was pre-inoculated into the soil, as compared to single inoculation experiments or experiments where Ag I

46

L. Krqelund.

0. Nybroe/

FEMS Microbiology

Ecology

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20 (1996)

41-51

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a 6 experiments are described: A-F. Experiments B, C, D, and E include two strains and are splitted up in two. * Significant differences between two experiments tested by r-test or, if the normality test or thwe equal variance test failed, by the Mann-Whitney Rank Sum test. Significant differences between two results are marked with the same * -number combinations. Experiments tested against each other were: A-B 1, A-C I, B I-C 1, Dl -E 1, C I -Dl ; F-B2, F-E2, B2-E2, D2-C2, E2-D2 and A-F, Bl-B2, Dl-D2. - = not measured.

F B2 E2 D2 c2

on on on in in

A BI Cl Dl El

seed seed seed soil soil

Inoculation method of Agl

Agl

Exp a

Table 2 Total P. ~7uorescens

and JMP134 were co-inoculated on the seed. The decrease in Agl cells was seen both at the root base and the tip. The soil-inoculated JMP134 established a population size that was higher, though not statistically significant, than that established after co-inoculation of both strains in the soil (Tables 1 and 2, exp. C2 and D2). So, JMP I34 pre-inoculated into the soil contribute to a change in the distribution of Agl and JMP134 by inhibiting Agl colonization. In the same way, the presence of pre-inoculated Agl in the soil had a profound effect on seed-inoculated JMPI 34 reducing its population significantly in all fractions at both the root base and root tip (Tables I and 2, exp. F and E2). The culturability of JMP134 in the fifth wash and in the homogenate was low as in the previous experiments (not shown). In these experiments, the Agl population was comparable to that established after co-inoculation of both strains in the soil (Tables I and 2, exp. D I and El).

Root

Root tie

base

The above experiments permit a comparison to be made between the distribution of soil-borne and seed-borne inoculants when established in the presence of a soil-borne competitor. Table I (exp. Cl and D I > shows that the population sizes of P. ,fluowscens Agl at the root base after either soil or seed inoculation into soil with JMP134 cells were not different from each other. At the root tip, however, (Table 2. exp. C I and DI) soil inoculation led to a significantly higher Agl population in the rhizosphere soil (6 X IO’ cells cm root- ‘J than inoculation via the seed (3 X IO’ cells cm root ‘>. In the reverse experiments, more A. eutrophus JMP134 cells were seen at the root base after seed inoculation (e.g. 3.5 X IO6 cells in the first wash) than after soil inoculation (8.7 X IO’ cells in the first wash) (Table I. exp. E2 and D2). This difference was most pronounced in the rhizosphere soil. At the root tip significant more A. eutrophus JMP I34 were present in rhizosphere soil if it had been introduced by soil inoculation (5 X IO” cells in the first wash) than by seed inoculation (3.1 X IO’ cells; Table 2. exp. E2 and D2). 3.5. &kcts of 2,4-D amendment

!. Washing 0

Agl

I

JMP

5.

horn.

fractions

134

Fig. 2. Seeds were inoculated with both P. ,fluore.ww~.\ Agl and A. rurrophus JMP134. 10 ppm 2,4-D was added to the soil. Bacteria from the rhizosphere (washes I. and 2.) and the rhizoplane (washes 5. wash and the homogenate) were harvested and the cell numbers were measured by immunofluorescence microscopy. Each data set represents the mean of results from four plants. Bars indicate standard errors.

2,4-D was added to the soil to see whether this amendment would change the competition pattern between Ag I and the 2,4-D degrader JMPI 34. Ag I and JMP134 were both introduced via the seed. Barley seedlings were able to develop from coated seeds in 2,4-D-treated soil, but the mean root length was only 2 cm. The distribution of Agl and JMP134 is shown in Fig. 2. As root length differed between this experiment and the corresponding experiment without 2,4D amendment (Tables 1 and 2, exp. B I and B2), the ratios between the Agl and JMP134 cell numbers are used to express the outcome of the competition. These ratios (Agl/JMPl34) were I .6, 2.3, 6.8 and 27.6 without 2,4-D, and 0.6, 1.5, 4.0 and 4.0 with 2.4-D amendment in fractions at the root base. The corresponding values from the root tip segments were: 2.9, 8.3, 26.0 and 28.0 versus 0. I, 0.1, I. I and 1.1.

4. Discussion 4. I. Distribution of P. jluorescens Agl und A. eutrophus JMPl34 in the rhizosphere In the absence of competition, seed-inoculated P. ,fluore.scens Ag I and A. eutrophus JMPI 34 establish comparable population sizes in the rhizosphere when total numbers are considered. Both strains are established on the entire root although cell numbers decline towards the tip. The ability to colonize the whole root is probably due to cell motility as water was not added during the experiments to provide passive transport [B-IO]. Previous results describing the distributions of cells between the rhizosphere and the rhizoplane are very inconsistent. We found in these studies comparable distributions for the two strains, the firmly attached cells accounting for ca. l-4% of the total population. Agl remains fully culturable in the barley rhizosphere whereas the subpopulation of A. eutrophus JMP134 closely associated to the root tip is nonculturable. This could be due to noxious substances, e.g. activated oxygen species, released from the proliferating plant cells found in this area [25]. Studies of bacterial population dynamics have often relied solely on culture methods. However, our results for JMPI 34 demonstrate that culture-dependent methods may provide misleading information about the distribution of bacteria in the rhizosphere. In these studies, without the presence of a competing indigenous microflora it is possible to determine the potential of the strains to associate with the developing root. Hence, the initial experiments demonstrated that the selected strains had properties considered important for colonization: to establish it on the entire root system [IO] and to associate it firmly to the root [5,16]. 4.2. Competitim

studies inlvll:ing

co-inoculation

In a round of competition assays, Ag 1 and JMP 134 were inoculated simultaneously either on the seed or in the soil. Simultaneous inoculation (no matter what inoculation method was chosen) had no effect on the ability of Agl to colonize the roots, whereas colonization by JMPI 34 was often reduced by the presence of Ag 1. Fluorescent pseudomonads are known

to be early colonizers able to utilize simple carbon sources present in exudates [17,26]. The superior root colonization by P. jluorescens Agl may therefore reflect its ability to respond to a greater range of substrates than JMP134 evident from the Biolog GN profiles of the strains. We found that addition of 10 ppm 2,4-D as a selective carbon source for JMP134 was able to improve its root-colonizing ability after co-inoculation with Ag 1. The herbicide 2,4-D has previously been used as a selective substrate for JMP134 in natural bulk soil by Jacobsen and Pedersen [ 191. They found that 10 ppm of 2,4-D did not result in any significant growth of JMP134 although the herbicide was rapidly mineralized. The different response to 10 ppm 2,4-D may be due to the large reservoir of 2,4-D in non-rhizosphere soil in our experiments as Jacobsen and Pedersen [19] found no mineralization of 2,4-D by the indigenous microflora. Thus, competition for substrate may be involved in determining the distribution of bacteria in the rhizosphere, as a poor competitor for substrate can be stimulated by addition of a selective carbon source. In this environment, it has previously been demonstrated that growth and activity of a salicylate-degrading pseudomonad could be enhanced by salicylate [27]. 4.3. Competition studies inlwll,ing mi.wed inoculation The presence of an established Agl inoculum in the soil reduced subsequent root colonization by seed-inoculated JMP134 in all fractions at both the root base and the root tip. However, an established JMP134 population in the soil was also able to reduce subsequent root colonization by seed-inoculated Agl. In these experiments we observed a changed distribution between the rhizosphere and the rhizoplane populations as JMP 134 primarily affected the rhizosphere population of Ag 1. This observation, and the low culturability of root-associated JMP134 at the root tip, suggest that JMP134 has a competitive disadvantage close to the root. This series of competition experiments imply that the presence of an established inoculum, even of a poor competitor for nutrients, may fill available niches and thereby interfere with the establishment

50

L. Kragelund,

0. Nybroe/

FEMS

of other organisms. Comparable observations have been made in bulk soil. Introducing an Arthrobacter strain to soil 21 days before adding a Flacobacterium strain significantly reduced the survival of Flacobacterium [28] compared to when the two strains were co-inoculated or the Flavobacterium was the sole inoculum. In natural soil it has been observed that seed-inoculated Pseudomonas strains did not colonize the lower part of the roots, whereas soil-inoculated cells colonized the whole root system [ 10,291. The authors concluded that transport of seed-coated organisms was important for dispersal. In contrast, we found comparable distributions of seed- and soil-inoculated bacteria along the root in a sterile system, but noted that root tip colonization by a seed-inoculated strain can be impaired by competition from pre-established bacteria in the soil. Consequently, competition may be just as important for the distribution of seed-borne inocula as active or passive transport. 5. Conclusions In conclusion, competition for limited carbon sources influence the outcome of root colonization, but even a poor competitor for nutrients can interfere with the establishment of other organisms, probably by filling available niches early during colonization. Root colonization of the root tip has previously been described to be dependent on active or passive transport of cells; however, these studies show that competition from pre-established bacteria in soil may be just as important.

Acknowledgements We would like to thank Lene Nielsen and MayBritt Prahm for technical assistance and Dr. Anne Winding for providing the Biolog Data. This work was supported by the Danish ‘Center for Microbial Ecology’. References [I]

Mazzola, pathogens

M. and Cook, R.J. (1991) Effects of fungal root on the population dynamics of biocontrol strains

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