Plant and Soil 153: 85-95, 1993. © 1993 Kluwer Academic Publishers. Printed in the Netherlands. PLSO 9902

Frankia in the rhizosphere of nonhost plants: A comparison with root- associated N2-fixing Enterobacter, Klebsiella and Pseudomonas

R. RONKKO 1, A. SMOLANDER 3, E.-L. NURMIAHO-LASSILA z and K. HAAHTELA l 1Department of General Microbiology, 2Department of Electron Microscopy, University of Helsinki, P.O. Box 41, SF-00014 Helsingin yliopisto, Finland and 3Department of Forest Ecology, Finnish Forest Research Institute, P.O. Box 18, SF-01301 Vantaa, Finland

Received 29 December 1992. Accepted in revised form 3 May 1993

Key words: Actinorhiza, Betula pendula, Betula pubescens, birch, Enterobacter agglomerans, Festuca rubra, Frankia, Klebsiella pneumoniae, nitrogen fixation, Poa pratensis, Pseudomonas sp., rhizosphere, root

Abstract

Bacterial growth in the rhizosphere and resulting changes in plant growth parameters were studied in small aseptic seedlings of birch (Betula pendula and B. pubescens) and grasses (Poa pratensis and Festuca rubra). The seedlings were inoculated with three Frankia strains (Aila and Ag5b isolated from native Alnus root nodules and Ai17 from a root nodule induced by soil originating from a Betula pendula stand), and three associative N2-fixing bacteria (Enterobacter agglornerans, Klebsiella pneumo- niae and Pseudornonas sp., isolated from grass roots). Microscopic observations showed that all the Frankia strains were able to colonize and grow on the root surface of the plants tested without addition of an exogenous carbon source. No net growth of the associative Nz-fixers was observed in the rhizosphere, although inoculum viable counts were maintained over the experimental period. Changes in both the biomass and morphology of plant seedlings in response to bacterial inoculation were recorded, which were more dependent on the plant species than on the bacterial strain.

Introduction

Although it has been shown that Frankia can survive and retain infectivity in soils devoid of actinorhizal plants (Arveby and Huss-Danell, 1988; Houwers and Akkermans, 1981; Huss- Danell and Frej, 1986; Smolander, 1990; Smolander and Sundman, 1987; Weber, 1986; van Dijk, 1984), the growth of Frankia has not been directly proven under these conditions. Surprisingly high densities of Frankia, deter- mined as nodulation units per soil volume, have been observed in soils under Betula pubescens (van Dijk, 1984), Betula pendula (Smolander, 1990) and Betula nigra (Paschke and Dawson, 1992). When development of the indigenous

Frankia population in a B. pendula soil was followed, an increase in nodulation capacity was observed which was most pronounced in soils planted with B. pendula seedlings (Smolander and Sarsa, 1990). Growth of Frankia in the rhizosphere of birch seedlings (B. pendula) was subsequently shown under laboratory conditions (Smolander et al., 1990).

Little is known about the survival and possible proliferation of Frankia in the rhizosphere of a nonhost plant or the surrounding soil. Other bacteria from several genera (e.g. Azospirillum, Enterobacter, Klebsiella and Pseudomonas) are known to live on plant roots as associative N z-

fixers. This suggests that Frankia may also sur- vive as an associative Nz-fixer, in a loose and

86 ROnkk6 et al.

unspecific relationship with the nonhost plant root. This hypothesis is supported by the fact that several Frankia strains are able to fix nitro- gen in pure cultures lacking any contact with their host plant. If the rhizosphere can offer essential nutrients, especially a suitable carbon source, in appropriate concentrations, N 2 fixa- tion should be possible. However, this kind of associative nitrogen fixation has not as yet been shown in the case of Frankia. In some studies of the interaction between associative Nz-fixers and plants, the importance of nitrogen fixation has been questioned while other factors were consid- ered more important (Bashan et al., 1989; Haahtela et al., 1988a; Okon et al., 1983; Smith et al., 1984). Plant hormones (Badenoch-Jones et al., 1982; Crozier et al., 1988; Ernstsen et al., 1987; Fallik et al., 1989; Haahtela et al., 1990) and siderophores (De Weger et al., 1986; Haahtela et al., 1990; Kloepper et al., 1980), which are produced by many plant-associated soil microbes, often have greater influence on plant growth than that resulting from N 2 fixation. In pure culture, Frankia strains have also been shown to produce plant hormones (Berry et al., 1989; Smolander et al., 1990; Stevens and Berry, 1988; Wheeler et al., 1984). Thus, it can be speculated that Frankia may grow in the rhizo- sphere as a saprophyte mainly obtaining carbon and energy sources from root exudates and dead root cells. Under these conditions Frankia may beneficially affect the root by producing plant hormones and possibly by protecting the root from pathogenic invasions.

In this study nonhost plant species (B. pen- dula, B. pubescens, Poa pratensis and Festuca

rubra) were inoculated with either different Frankia strains (isolated from native nodules or from nodules induced by soil from a B. pendula stand) or a range of associative Nz-fixers (En- terobacter agglomerans, Klebsiella pneumoniae and Pseudomonas sp.) which had been isolated from grass roots. The growth of bacteria in the rhizosphere and the effect of these bacteria on different plant growth parameters are described and discussed.

Material and methods

Bacterial strains

Bacterial strains, including both Frankia strains and associative N2-fixers, are listed in Table 1. The Frankia strains were cultivated in static cultures in PC medium (Smolander and Sarsa, 1990), pH 6.8 at 28°C, collected, homogenized and quantified using a protein assay as described earlier (Smolander et al., 1988b). The inoculum was diluted with full strength HD (mineral medium) which contained 1 mM KNO 3 as a sole nitrogen source and no carbon source (Huss- Danell, 1978; the amount of KzHPO 4 and KH2PO 4 was doubled in order to increase buf- fering capacity). The associative N2-fixers (Kleb- siella, Enterobacter and Pseudomonas strains) were grown prior to inoculation in static malate medium cultures for 48h at 28°C (Haahtela et al., 1983; Korhonen et al., 1983). Cell densities were estimated microscopically (Petroff-Hauser chamber) and adjusted to 8 x 107 for all strains and a viable count was made of each inoculum.

Table 1. Bacterial strains and their origin

Strain Origin" Reference

Actinorhizal N 2-fixers Frankia Aila (UHF 01111 b) Frankia Ail7 Frankia Ag5b

Associative N2-fixers Enterobacter agglomerans Am (EaAm) Klebsiella pneurnoniae As (KpAs) Pseudomonas sp. Dc (PsDc)

Alnus incana Betula pendula stand Alnus glutinosa

Achillea millefolium Agrostis stolonifera Deschampsia caespitosa

Weber et al., 1988 Smolander and Sarsa, 1990 Weber et al., 1988

Haahtela et al., 1981, 1983 Haahtela et al., 1981, 1983 Haahtela et al., 1981, 1983

aFrankia Aila and Ag5b were isolated from native nodules and Ait7 from A. incana nodules induced by a soil inoculum; the associative Nz-fixers were isolated from the rhizoplane. bFrankia catalogue number (Lechevalier, 1985).

Frankia in the rhizosphere of nonhost plants 87

Plant material and inoculation

Seeds of P. pratensis and F. rubra were surface sterilized by stirring treatment with 94% ethanol for I min followed by 5% (w/v) sodium hypo- chlorite for 12min and washed five times in sterile water (Korhonen et al., 1983). Seeds of B. pendula and B. pubescens were surface steril- ized in 30% H202, supplemented with a few drops of Tween 80, for 25 min and washed as above (Smolander et al., 1990). All seeds were allowed to germinate in the dark on 0.7% glucose - mineral nutrient agar (K2HPO4, 7gL-1 ; KH2PO4, 2 g L - l ; MgSO4.7H20 , 0 .2gL-1; (NH4)2SO4, l g L - 1 ; agar, 10gL -1) at room temperature for 5-7 days.

Seedlings from plates without any sign of contamination were transferred to sterile glass test tubes (22mm diameter, 200mm high) capped with cotton plugs. The tubes contained 15 mL of glass beads (3 mm diameter) moistened with 7.5 mL of full strength HD medium (see earlier). Seedlings were grown at 24 -+ 2°C with a photoperiod of 18h. The light source was fluorescent daylight tubes (Daylight 5000 Special Deluxe, 36W, Airam, Finland) giving a fluence rate of 50/zMol m -2 s -~ (Quantum Photometer LI 185 A, Lambda Inst. Corp., USA). Four weeks after the sterilization, seedlings of approx- imately equal size were inoculated with either a 0.5 mL inoculum of Enterobacter, Klebsiella or Pseudomonas adjusted to contain about 8 × 107 cells, or a 1 ml inoculum of Frankia (in HD), containing 1, 2, 5, 10 or 25/.~g of Frankia cell protein. The initial quantity of Frankia was visually estimated on the root surface of a few inoculated plants under the light microscope.

During the incubation period HD and sterile water were added to the tubes to compensate for evaporation and to maintain the original pH (about 1 mL of HD was added per tube weekly). A total of 9-10 and 3-4 replicate plants were respectively subjected to plant growth and light microscopy and for electron microscopy and viable count analysis. Equal numbers of plants, supplemented with a respective volume of bac- teria-free HD were treated as controls in each experiment. The experiments shown here were preceded by preliminary tests with varying in- oculum sizes and incubation periods.

Analysis of plant growth parameters and bacterial growth

Plants were removed from the tubes 5-8 weeks after inoculation (9-12 weeks after seed steriliza- tion), and the following growth parameters were measured: dry and fresh weight (root and shoot), length (root and shoot), leaf area (Betula), leaf length (Poa and Festuca), abundance of root hairs (microscopically estimated) and number of lateral roots. Dry weight was measured after 18 h at 70°C (dry weights of 12-week-old seedlings were of the magnitude of 25 mg). Leaf area of birch was measured by a scanner or video digitizer connected to a microcomputer and by performing image analysis of the digitized pic- tures. The sum of the leaf lengths was used as an estimate of leaf area for Poa and Festuca. Number of lateral roots (number of root tips) was counted (branches t>0.5 mm long) on an illuminated table (lateral root numbers of 12- week-old seedlings were of the order of 100). The pH of the culture medium was measured.

Growth of Frankia in the rhizosphere follow- ing inoculation, i.e. the increase of the initial inoculum, was visually estimated by light and scanning electron microscopy (SEM). For light microscopy the roots were gently separated from the glass beads, examined and photographed. Abundance was compared to that after 1-2 days from inoculation. SEM was performed as de- scribed earlier (Smolander et al., 1988a). Viable count of associative N2-fixers attached to the surface of roots was measured as described by Haahtela et al. (1988a). Viable count of bacteria in the culture liquid was assayed by removal of the liquid from the tubes under vacuum, diluting and plating.

Results

Growth of Frankia in the rhizosphere

Examination of roots by light and scanning electron microscope revealed that all three Fran- kia strains were able to grow in the rhizosphere (or rhizoplane) of all of the tested plants (B. pendula, B. pubescens, F. rubra and P. pratensis) without an added carbon source. (Table 2). In

88 ROnkk6 et al.

Table 2. Growth of Frankia strains Aila, Ail7, and Ag5b in the rhizosphere of non-host plants without an additional carbon source

Frankia strain Plant species

B. pendula B. pubescens F. rubra P. pratensis

Aila + + + + + ++ + ++ Ail7 (+) (+) + + Ag5b + + + + + + + +

Growth was observed microscopically and classified from weak (+) to strong + ++, as compared to reference plants microscoped 1-2 days after inoculation. Incubation time was 6-8 weeks and inoculum size 1-5/zg of Frankia protein.

the rhizosphere of B. pendula and B. pubescens growth of A i l a and Ag5b was similar and more vigorous than that of Ai l7 (1 p~g inoculum). In the rhizosphere of F. rubra and P. pratensis growth of A i l a was most abundant, and growth of Ag5b was better than that of Ai l7 ( 2 - 5 / z g inoculum). Figure 1 shows the colonization and growth of Ag5b on both root hairs and the root surface of F. rubra.

The Ai l7 and A i l a hyphae regularly contained vesicles, which contrasted to the rare occurrence in Ag5b. In the case of Ai l7 vesicle formation was abundant, which is also typical for this strain in pure culture. Sporangia were regularly ob- served in Ai l7 , whereas in A i l a they were more rarely seen and in Ag5b they were almost ab- sent. The presence of vesicles and sporangia was not dependent on the plant species colonized.

The abundance of Frankia was also found to be proportional to the inoculum size. Frankia was usually abundant but restricted to the rhizo- plane following growth from a small inoculum (1/xg) but when a larger inoculum was used (5 and 25/~g) dense and large Frankia 'mats ' and clusters could be seen both on the rhizoplane and in the medium. In static pure cultures (PC medium) the growth rates of Ai la and Ai l7 are approximately equal, whereas Ag5b grows con-

siderably slower and less vigorously than the others. This pattern was changed in the rhizo- sphere, where Ag5b grew more and Ai l7 less vigorously than in pure cultures (Table 2).

Growth o f Klebsiella, Enterobacter and Pseudomonas in the rhizosphere

B. pendula seedlings were inoculated with K. pneumoniae, E. agglomerans and Pseudomonas sp. After 8 weeks the viable count of bacteria attached to root and unattached in the medium was assayed (Table 3). The number of viable cells in the E. agglomerans inoculum was similar to the total cell number estimated microscopical- ly, but in the K. pneumoniae and Pseudomonas sp. inocula the proportion of non-growing cells was considerable (60%). In the rhizosphere of B. pendula, the total number of living En- terobacter and Pseudomonas cells was almost the same as in the initial inoculum, but the viable count of K. pneumoniae had decreased to one third of the inoculum (Table 3). The number of root-attached bacteria (rhizoplane) was propor- tional to the number of living cells in the in- oculum.

The attachment of associative N2-fixers to F. rubra roots was also assayed, and the results

Table 3. Cell numbers of E. agglomerans, K. pneumoniae, and Pseudomonas sp. in the inoculum and in the rhizosphere of B. pendula 8 weeks after inoculation (x 10 6 cfu/plant) a

Strain Inoculum size V.C. b of bacteria in

Microscopy c V.C. rhizoplane medium total

E. agglomerans Am 81 86 14 63 77 K. pneumoniae As 81 29 2.1 7.5 10 Pseudomonas sp. Dc 80 33 4.2 26 30

aAll numbers are mean of four replicates; cfu, colony forming units. bViable count. CPetroff-Hauser chamber.

Frankia in the rhizosphere of nonhost plants 89

Fig. f. Frankia AgSb in the rhizosphere of F. rubra 5 weeks after inoculation. Inoculum size was 10 ,ug of Frankia protein per plant. Frankia colonized primarily the root hairs (A), but also sporadically the root surface (B). The bar represents 10 pm in A and 20 pm in B.

9 0 R 6 n k k 6 et al.

A) ROOT DRY WEIGHT

lOO 1 . , 90

80

g 70

~ 60 so i ) ( .~ ) . ,

,o I:m 30 (*)

" O t poSou, i B. ) P. ) . . . i pubescensi pratensis i pendula -20

Aila Ai17 Ag5b EaAm KpAs PsDc -4 [] • • • • •

D) LATERAL ROOT NUMBER / ROOT LENGTH 8O .¢: 70 ¢:

e 60

E so

~ 40

30 20

~ 10, N [o -10,

g -2o "~ -30,

-40, • ii'F.

pendula )p " si P tensis rubra

(*) i

ii, Aila Ai17 Ag5b EaAm I ~ PsDc -4 • • • • • •

5O

40

3o

20

~ 10

"~ 0

g -lo .5

-2o -30

-40

-50

B) ROOT LENGTH

dula pub~ S prMPensls

Aila Ai17 AgSb

F. rubra

mm- m _ .

B. F. pendula rubra

EaAm KpAs PsDc-4

4 0

30.

20-

10.

E o

-10.

"~ "20- "6 -3o-

¢~ -40-

"SO

"6C

E) LATERAL ROOT NUMBER / ROOT DW

J (,) * *

~F1 Ho uH , .

Aila A i17 Ag5b Ea/~n Kpks PsDc -4 [ ] • • • • •

C) LATERAL ROOT NUMBER 80 70 i

50 ~ 40

30 **

"6 10

g -lo "~ -20

-3o B. i 8. i P. i F. "40 pendula ipubescens! pratensis i rubra

Aila Ai17 Ag5b

D • •

**= !

s

i m.i B. i E

~endula i rubra

Kp~ PsDc

50.

40.

30.

• ~ 20-

.~ lo. "6 0. 8, ,~ t o . -8o -20. o~ -30-

-40-

-50

F) SHOOT DRY WEI(

(,)(*) i

B. B. :: P. E pendula ipubescensi pratensis ! rubra

IHT

=

B. F. pendula rubra

Aila A i17 Ag5b [ ] • •

EaAm KpAs PsDc-I [ ] • •

Frankia in the rhizosphere of nonhost plants 91

50.

40.

3 0

_.¢ 2 0

• .6 0

~ -10 C

g -2o

N -30

-40

-513

G) SHOOT LENGTH

°

) i i

B. i B. ) P. i F. pendula ipubescens i pratensis i rubra

I !!"

B. F. pendula rubra

Aila Ai17 Ag5b [ ] [ ] [ ]

EaAm KpAs PsDc-4

I) PLANT DRY WEIGHT

50-

40 - (,) : **,

-m 20- :

° ! H 0 17 N E1 ) -- m

° )==i '"° ,m - t o i

"~ -20

-30

- 4 0 B. : B. !i P. i F . B. F . "50 pendula ~pubescens i pratensis i rubra ~endula ~ rubra

Aila Ai17 Ag5b EaAm KpAs PsDc--I [] [] [ ] [ ] • •

100

80

t~ 60

---¢ 40 "6 O ~ 20

g o

-20-

"4£

H) LEAF AREA

PI l l I-I ll U,~

B. B. P. F. pendula ipubescensi pratensis rubra

.i !1"

B. E pendula rubra

140

120 O

100

o 80

"5 60 0

"6 40 (11 ~ 20

-20

-4C

J) ROOT / SHOOT RATIO

u + B

B. pendula

i i

z

~ o. ! p. ) F. )pubesc~s) pratensis ) rubra

)

BI :: F. pendula ) rubra

Aila At17 Ag5b EaAm KpAs PsDc Aila At17 Ag5b Ea/tzn KpAs PsDc [] [] [] [] • • D [] [] [] • •

Fig. 2. Effect of Frankia (Ai la , A i l 7 and Ag5b) and associative Nz-fixers ( E a A m , KpAs and PsDc) on growth parameters of B. pendula, B. pubescens, P. pratensis and F. rubra seedlings. The effects on each parameter (A-J) are shown as a percentage change compared to uninoculated control plants (mean of 9-10 replicate plants). (*), *, ** and *** show the statistical significance of the difference in t-test at 10, 5, 1 and 0.1% significance levels respectively. Inoculum size of Frankia was quantified as cell protein and was 1-5/zg/p lant . Inoculum size of associative N2-fixers varied from 29 × 106 to 86 × 106 cfu/plant for B. pendula and from 0.44 × 106 to 145 × 106 cfu/p lant for F. rubra. Seedlings were inoculated 4 weeks after germinat ion and incubated with bacteria for 8 weeks (B. pubescens for 6 weeks). Dw, dry weight.

(data not shown) were similar to those observed by Haahtela et al. (1988a). In contrast to B. pendula (Table 3), the root-attached population was not proport ional to the inoculum size.

Effect of bacteria on plant growth parameters

Plants were inoculated with Frankia or associa- tive Nz-fixing strains. After 6 -8 weeks the plant growth parameters were measured and the fol- lowing ratios were also calculated from the primary growth parameters: lateral root number per root length and per root dry weight, and

root / shoot ratio (dry weight /dry weight). The percentage change compared to sterile control plants was calculated for each parameter . The fresh weight values (root, shoot, and whole plant) paralleled the respective dry weights and are not shown. The root hairs were abundant with all plant species and no visible differences were observed between inoculated and uninocu- lated plants.

The influence of Frankia and associative N 2- fixers on root biomass and morphology are shown in Fig. 2A-E. Overall, the bacterial inoculi tended to increase root biomass (dry

92 R6nkk6 et al.

weight, Fig. 2A). Frankia strains induced a not- able increase of root biomass in grasses, especial- ly in P. pratensis, whereas the effect on birch roots was negligible. All the associative N2-fixers increased root biomass of F. rubra significantly, and the most effective strain, EaAm, also that of B. pendula.

The influence of bacteria on root morphology was more variable. The root length of grasses tended to increase both with Frankia and as- sociative Nz-fixers, whereas in birches significant decreases of this parameter was observed with some Frankia inoculi (Fig. 2B). The lateral root number of B. pendula and P. pratensis increased with all inoculi, whereas the effect on B. pubes- cens and F. rubra was less abundant (Fig. 2C). When lateral root number was calculated per root length (Fig. 2D) and dry weight (Fig. 2E), the birches and grasses differed distinctly from each other, the birches showing in most cases an increase and the grasses a decrease of relative lateral root number. Most clearly this change was seen in B. pendula and F. rubra inoculated with associative Nz-fixers (Fig. 2E).

The influence of Frankia and associative N 2- fixers on shoot biomass and morphology are shown in Fig. 2F-H. In overall, the biomass of both B. pendula and P. pratensis tended to increase with all inoculi, whereas in F. rubra some decrease was observed (Fig. 2F). The changes in shoot length were not very pro- nounced, but Frankia seemed to decrease this parameter in birches and increase it in F. rubra (Fig. 2G). Interestingly, the effect of associative Nz-fixers on shoot length of F. rubra and B pendula tended to be opposite to that of Frankia on the same species. The effect of Frankia on plant leaf area was relatively small ( < 1 5 % ) and in most cases insignificant (Fig. 2H). This was contrasted by the drastic increase (100%) seen on B. pendula inoculated with EaAm, and to a lesser extent with PsDc. On F. rubra the effect of associative Nz-fixers was a decrease and less pronounced.

The influence of Frankia and associative N z-

fixers on the biomass of whole seedlings is shown in Fig. 2I-J. In most cases Frankia and associa- tive Nz-fixers did not notably affect the biomass of seedlings, but significant increases were ob- served in P. pratensis inoculated with Frankia

Aila and Ail7, and in B. pendula inoculated with EaAm and PsDc (Fig. 2I). The biomass distribution between root and shoot ( root /shoot ratio) was not notably changed in Betula species, but significant increases were observed in F. rubra both with Frankia Ai la and the associative Nz-fixers (Fig. 2J). EaAm and KpAs strains were especially effective, resulting in a very dramatic increase ( > 100%) in the ratio.

The pH of the medium was measured after all plant experiments. The original pH of the medium (6.0) rose in all experiments (min 6.3, max 7.0). The difference in pH between inocu- lated and uninoculated tubes was in most experi- ments negligible. However , when B. pendula was grown with E. agglomerans, K. pneumoniae and Pseudomonas sp., the pH was significantly higher (about 0.3 pH units), and when B. pubes- cens was grown with Frankia Aila , significantly lower (0 .2pH units) than that of uninoculated controls.

Discussion

Our experiments showed that three different Frankia strains originating from two Alnus species and a B. pendula stand were able to colonize and grow in the rhizosphere of both Betula and grass species. These results suggest that the root exudates of all plant species studied contain suitable carbon and energy sources that can support growth of Frankia. Root exudates have already been shown to contain a great variety of substances, including organic carbon sources (Smith, 1976). Interactions between Frankia and grasses (or other annual nonhost plant species) in natural situations have not been reported, but this possibility must be born in mind when explanations for the high Frankia densities in sites devoid of host plants are consid- ered. Electron micrographs (Fig. 1) also sug- gested some form of at tachment of Frankia both to the root hairs and the root surface of F. rubra but further studies are needed to confirm this observation and to reveal the mechanism and specificity level.

It is clear that microscopical methods only allow the study of the root-colonizing fraction of Frankia and thus other methods are needed to

Frankia in the rhizosphere of nonhost plants 93

estimate the growth more precisely, including the Frankia population in the medium. Our preliminary findings suggest that filtration and protein assay can be used to separate and quan- tify Frankia in the medium, due to the low plant protein background and use of easily separable support material (glass beads). However , in the case where small inoculum sizes are used the sensitivity of the protein assay and separation of Frankia from the root surface are problematic. Other biochemical methods, which are more specific for bacteria (e.g. muramic acid or diaminopimelic acid assay), are promising alter- natives for quantification of Frankia in the rhizo- sphere.

In studies on the interaction and attachment of K. pneumoniae As, E. agglomerans Am and Pseudomonas sp. Dc to the roots of P. pratensis (Haahte la et al., 1986, 1988a, b; Haahtela and Korhonen , 1985) viable counts of bacteria at- tached to roots but not numbers of bacteria in the medium were assayed. Although we ob- served no net growth of these strains in the rhizosphere of B. pendula, the birch seedlings were able to maintain the viability of the in- oculum at the original level. The size of the root-at tached bacterial population was to some extent proport ional to the initial inoculum size (viable cells), in contrast to our results with F. rubra and earlier studies with P. pratensis (Haahte la et al., 1988a), where no correlation was seen.

In the inoculation experiments with different birch and grass species, significant changes in plant morphology and /o r biomass were induced by both Frankia and associative N2-fixers. The directional change of plant growth parameters seemed to be mostly dependent on the plant species and to a lesser extent on the bacterial group (Frankia versus associative N2-fixers ). Within both of the bacterial groups the direction- al change was mainly the same for all strains, but notable quantitative differences were observed between strains. In one experiment where B. pendula had been exposed to Ai l7 somewhat contradictory results were obtained (lateral root number and leaf area) to those shown in Figure 2. Whether this was due to differences in the condition of the inoculum or seedlings is not known.

The root / shoot ratio of both birch species remained essentially unchanged in all experi- ments, even in the case where there was a significant change in the total biomass, whereas in F. rubra exposed to associative N2-fixers and Frankia Ai la considerably higher values were obtained. Unlike in F. rubra, the increase in lateral root numbers was the most common and usually the most notable change in birch. The two grass species, F. rubra and P. pratensis, showed somewhat different response patterns when inoculated with Frankia. Interestingly, E. agglomerans (originating from grass roots) had a very strong positive effect on B. pendula seed- lings (Fig. 2), in particular a doubling of leaf area. Practically nothing is known about the bacterial population colonizing birch roots in nature, and thus the value of this observation remains unclear.

The exact mechanism of these bacterially induced changes in the seedlings is not yet known but plant hormones are the most prob- able candidates. All of the bacterial strains used in this work are able, in an appropriate medium, to produce indole compounds which are known to express plant hormonal activity. Indole-3- ethanol has been shown to be produced by the Frankia strains (Smolander et al., 1990), where- as the associative N2-fixers are capable of producing indole-3-acetic acid (auxin), indole-3- ethanol and several other indoles (Haahtela et al., 1990). The production of compounds belong- ing to other plant hormone groups (e.g. gibberel- lins and cytokinins) has not been studied in these strains. It should be noted here that indole-3- ethanol is known to induce lateral root primordia (Berry et al., 1989), being thus a very likely reason to the increase in lateral root number observed in some of our experiments.

As already noted, an abundant Frankia popu- lation (nodulation capacity) was found in Finnish birch soils (Smolander 1990) and planting of birch seedlings increased the nodulation capacity of these soils (Smolander and Sarsa, 1990). The inevitable question is whether there is any specificity in the interaction between birch and Frankia which could possibly give Frankia an advantage over other microbes in the rhizo- sphere. In our inoculation experiments the as- sociative N2-fixers were able to both colonize the

94 R6nkk6 et al.

root of B. pendula and affect the plant growth parameters but, in contrast to Frankia, no growth was observed in the rhizosphere. How- ever, due to the different quantification meth- ods, the final conclusion about specificity can not as yet be made. It is obvious that in natural conditions a mixture of different microbes in- stead of a single strain is present in the rhizo- sphere, and thus coinoculation experiments would be needed to answer this question. Another fascinating but still unanswered ques- tion is, considering the close relatedness of Betula and Alnus (Bousquet et al., 1989), whether there are some events or signals in the early phases of actinorhizal symbiosis that are common in the interaction between Frankia and birch species.

Acknowledgements

We are grateful to Prof. Veronica Sundman and Dr. Robin Sen for constructive comments on the manuscript and to Yvonne Bj6rkman for techni- cal assistance. The language was revised by Dr. Robin Sen. This work was supported by the Foundat ion for Research of Natural Resources in Finland.

References

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Section editor: R 0 D Dixon