The effect of Frankia and Paxillus involutus on theperformance of Alnus incana subsp. rugosa inmine tailings

John H. Markham

Abstract: The purpose of this study was to determine whether symbiotic nitrogen-fixing bacteria and mycorrhizal fungiact synergistically to improve plant performance when grown on heavy metal mine tailings. Seedlings were inoculatedwith Frankia, Paxillus involutus (Batsch) Fr., or a combination of both and grown in 100% peat, a 1:1 mix of peat andtailings, or 100% tailings for 20 weeks. Mortality of plants grown on pure tailings (15.0%) and peat–tailings (17.9%) wassignificantly greater than mortality of plants grown on peat (3.5%). The rate of nodulation and mycorrhizae formation de-creased from 90.0% and 66.7%, respectively, on the peat, to 11.9% and 2.6% on the tailings. Frankia-inoculated plantsgrown on peat–tailings showed twice the mortality rate (38.5%) of any other inoculation treatment. Plants grown on mediacontaining tailings had greater root/shoot ratios than plants grown on peat. Inoculating plants grown in the presence of tail-ings with either Frankia and (or) P. involutus increased root thickness. Inoculating plants with both symbionts increasedcolonization rates and shoot yield on the peat and peat–tailings media, suggesting that these symbionts act synergisticallyto improve plant performance. However, inoculating plants with Frankia decreased shoot relative growth rate in the earlypart of the experiment when plants were not fixing nitrogen, which, coupled with the higher mortality effect, suggests thatnodule development is a stress for plants. It may be advisable that plants have fully functioning nodules before transplant-ing if they are to be used in revegetation programs.

Key words: Alnus incana subsp. rugosa, Frankia, Paxillus involutus, mine tailings, mycorrhizae.

Resume : Le but de l’etude etait de determiner si les bacteries fixatrices d’azote et les champignons mycorhiziens peuventagir en symbiose, pour ameliorer la performance des plantes, lorsqu’on les cultive sur des rejets de mines de metaux.L’auteur a inocule des plantules avec le Frankia, le Paxillus involutus (Batsch) Fr., ou une combinaison des deux, et les acultivees soit dans le la tourbe, dans un melange 1:1 tourbe et rejets, ou dans 100 % de rejets, pendant 20 semaines. Lamortalite des plants cultives dans les rejets purs (15,0 %) et le melange tourbe–rejets (17,9 %), est significativement pluselevee que la mortalite des plants cultives dans la tourbe (3,5 %). Les taux de nodulation et de mycorhization diminuent de90,0 % et 66,7 %, respectivement, dans la tourbe, a 11,9 % et 2,6 % dans les rejets. Les plantes inoculees avec le Frankiaet cultivees sur tourbe–rejets montrent un taux de mortalite deux fois plus eleve (38,5 %) que tous les autres traitementsd’inoculation. Les plantes cultivees sur des milieux contenant des rejets montrent des rapports racine/tige plus grands queles plantes cultivees dans la tourbe. L’inoculation des plants avec le Frankia et (ou) le P. involutus augmente l’epaisseurdes racines. L’inoculation des plants avec les deux symbiontes augmente les taux de colonization et le rendement des tiges,dans les milieux de tourbe et de tourbe–rejets, ce qui suggere que les deux symbiontes agissent en synergie pour ameliorerla performance de la plante. Cependant, l’inoculation des plantes avec le Frankia diminue le taux de croissance relative audebut des experiences, quand les plants ne fixent pas encore l’azote; ceci, couple avec une forte mortalite, suggere que ledeveloppement des nodules constitue un stress pour la plante. Consequemment, il y aurait avantage a ce que les plantsportent des nodules totalement fonctionnels avant de les transplanter, lorsqu’ils sont destines a des programmes de rehabi-litation de la vegetation.

Mots cles : Alnus incana subsp. rugosa, Frankia, Paxillus involutus, rejets de mines, mycorhizes.

[Traduit par la Redaction]

Introduction

Soil microbial symbionts are known to increase the per-formance of plants. In the case of mycorrhizal fungi, this in-creased performance is attributed to increasing the plants’access primarily to phosphorus (Smith and Read 1997),

whereas symbiotic nitrogen-fixing bacteria increase plant ac-cess to nitrogen. It is also known that mycorrhizae and sym-biotic nitrogen-fixing bacteria can act synergistically,increasing plant growth and rates of nitrogen fixation whenboth are present, compared with the presence of a singlesymbiont. While there is extensive research showing syner-gistic effects between legumes, rhizobial bacteria, and my-corrhizal fungi (Barea and Azcon-Aguilar 1983; Smith andRead 1997), much less work has been done on the actinorhi-zal plant Frankia and mycorrhizal fungi systems (but seeTian et al. 2002; Chatarpaul et al. 1989).

Establishing plant populations in poor soil conditions re-

Received 22 July 2005. Published on the NRC Research PressWeb site at http://canjbot.nrc.ca on 11 January 2006.

J.H. Markham. Botany Department, University of Manitoba,Winnipeg, MN R3T 2N2, Canada (e-mail:markhamj@cc.umanitoba.ca).

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mains a challenge. Perry et al. (1989) suggested that a posi-tive feedback can occur between the plant and the soil mi-crobial communities. Each community supports anddepends on the other such that, when one community iseliminated from a site by some form of disturbance, theother community will be negatively affected. On sites wherethe environmental conditions are stressful, because of eitherlow resource availability or toxic conditions, it may becomedifficult to reestablish a plant community without introduc-ing a soil microbe community at the same time. This feed-back between the plant and soil microbe community mayexplain Grubb’s (1983) observation that plants that formsymbiotic nitrogen-fixation relationships are rarely found asinitial colonizers during primary succession. It may be thatsoil resource availability under early successional conditionsis insufficient for either the bacteria or the host plant to existindependently for a long enough period so that the symbioticrelationships can become established.

Abandoned mine tailing sites may represent situationswhere revegetation is difficult because of the interdepend-ence of the plant and soil microbe community. Mineral ex-traction associated with hard-rock mining is oftenaccompanied by the production of fine tailings from thecrushing of rock. These materials are usually left on site inwhat are referred to as tailings ponds and may be manymetres thick and many hectares in area. The tailings tend tohave little organic matter and available nitrogen, generallydevelop little structure, and therefore have poor aerationand high bulk density (Bagatto and Shorthouse 1999; Levyet al. 1999). They may also have high heavy metal concen-trations, creating toxic conditions both for plants and soilmicrobes (Rajapaksha et al. 2004). As such, tailings pondsoften remain unvegetated for extended periods.

The hypothesis for this study was that inoculating plantswith combinations of two types of microbial symbionts, ec-tomycorrhizal fungi and nitrogen-fixing bacteria, would im-prove plant growth and survival on mine tailings. Alnusincana subsp. rugosa (Du Roi) K. Spreng. (speckled alder)was chosen as the test plant species, since it forms symbioticrelationships with nitrogen-fixing bacteria in the genusFrankia and with a limited number of mycorrhizal fungi, in-cluding Paxillus involutus (Batsch) Fr. (Godbout and Fortin1983). The ability of alders to form nitrogen-fixing symbio-ses can result in sites with alders undergoing substantial in-creases in soil nitrogen and organic matter (Crocker andMajor 1955). They can therefore act as species that facilitatenatural succession and site restoration (Aber 1987; Brad-shaw 1983).

Materials and methods

The tailings used in the study were produced by the Cen-tral Manitoba Mine (50854’16.6@N, 95820’12.2@W) that oper-ated from 1927 to 1937 (Slivitzky 1996). The tailings pondis approximately 20 ha. Almost no plants occupy the tailingspond. Near the edge of the tailings pond, some scatteredPoa pratensis L. and stunted Populus balsamifera L. can befound, but aerial photographs from 1934 and 1986 show nochange in the extent of the tailings pond or vegetation cover.The tailings have high concentrations of available heavymetals as determined by inductively coupled plasma – mass

spectrometry (ICP-MS) of ammonium iodide extractions. Inthe upper 30 cm of tailings, the highest heavy metal concen-tration is copper, at 718 ppm (Renault et al. 2000). Othersignificant heavy metal concentrations (in ppm) include Mn,40; Bi, 11; Br, 7; and Zn, 4. The pH of the tailings is highlyvariable throughout the site and through time (S. Renault,personal communication). The tailings that were collectedhad a pH of 7.76, as measured in a 1:1 0.01 mol�L–1 CaCl2solution. The tailings are low in inorganic N and available P(see below).

An initial assay was conducted to determine the inocula-tion potential of the tailings. A total of 30 Alnus crispa(Ait.) Pursh seedlings grown from a local seed source weretransplanted to pots containing sterile Turface1 and approx-imately 1% tailings by volume (a homogenized sample oftailings used in the inoculation experiment). After growingthese plants for 6 weeks in a greenhouse, their roots wereexamined as described below. None of the plants developedectomycorrhizal mantles or Hartig nets. Two plants devel-oped nitrogen-fixing nodules, indicating a low inoculationpotential for both types of symbionts in the tailings.

For the inoculation experiment, tailings were collectedfrom the top 30 cm of an area approximately 10 m � 10 m.The tailings were air dried, mixed, and stored in sealed con-tainers until used. Alnus incana subsp. rugosa seeds werecollected from a number of uncontaminated stands in south-eastern Manitoba in the fall of 2002. Seeds were germinatedon sterile 50% Turface1 : 50% vermiculite potting mix. In-dividual seedlings were transplanted to small pots once thefirst true leaves were produced. They were fertilized twicewith a commercial hydroponic nutrient solution (3.3 mL�L–1

FloraGro and 1.7 mL�L–1 FloraMicro, General Hydroponics,San Rafael, California) containing (in mmol�L–1) N, 9.3; P,1.1; and K, 4.6. Inoculation took place 4 weeks after thelast fertilizer application.

Seedlings received one of the following inoculation treat-ments: (i) control, that is, no inoculation; (ii) inoculationwith Frankia; (iii) inoculation with P. involutus; (iv) inocu-lation with both Frankia and P. involutus. The Frankia inoc-ulum was made from a collection of A. incana subsp. rugosanodules collected off the mine site, approximately 5 kmfrom the tailings pond. The nodules were washed, surfacesterilized with 30% H2O2 for 1 min, and then rinsed threetimes in sterile distilled water. The nodules were crushed ina sterile mortar and pestle and suspended to a concentrationof 2 mg�mL–1 in TEA buffer (in mmol�L–1: tris–HCl, 10;Na-EDTA, 1; ascorbic acid, 20; pH 7.6) supplemented with2% polyvinylpyrrolidone (PVP). Each plant was inoculatedwith 1 mL of the suspension at 12 weeks after germination.The P. involutus inoculum was from a culture (accessionNo. 5817) from the University of Alberta Mycological Her-barium. The culture was grown on potato dextrose agar(PDA) then scraped from the plates and mixed in a sterilemortar and pestle with PBS buffer (in mmol�L–1: KH2PO4,1.4; K2HPO4, 4.3; NaCl, 137; pH 7.3). An aliquot of thesuspension was centrifuged to determine the packed cell vol-ume, and 50 mL packed cell volume was injected in therooting zone of each plant. Plants were inoculated at13 weeks after germination.

One week following the P. involutus inoculations, plantswere transplanted to 5 cm diameter � 18 cm deep contain-

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ers (Ray Leach Deepots, Stuewe & Sons Inc., Corvallis, Or-egon) containing 100% horticultural peat, a 50% peat : 50%tailings mix (by volume), or 100% tailings. There were 20plants per combination of inoculation and planting media.The peat was stored in a drying oven at 70 8C for 3 weeksprior to transplanting to reduce fungal populations. The levelof P, as determined from NH4F extracts (Kalra and Maynard1991), was 2.55 mmol�L–1 in the peat, 0.66 mmol�L–1 in thepeat–tailings, and 0.00 mg�kg–1 in the tailings. Total inor-ganic N, as determined from 2 mol�L–1 KCl extracts andsteam distillation (Mulvaney 1996), was (in mmol�L–1)1.00 in the peat, 0.66 in the peat–tailings, and 0.16 in thetailings. Sterile clay pellets were placed in the bottom ofthe containers up to the level of the drainage holes beforefilling the containers with planting medium. Each containerwas assigned a random position on the greenhouse bench,and the racks in which the containers were held were rotatedon the greenhouse bench twice weekly. The plants weregrown under supplemental metal halide lighting (providingan additional approximately 200 W�m–2 at bench level for16 h�d–1) and watered daily to field capacity using an auto-matic overhead misting system. The watering period was ad-justed periodically to minimize the amount of water drippingout of the pots and the amount of salt crusting on the surfaceof the tailings media. Nitrogen fixation was monitored on arandom subsample of plants 6 and 12 weeks after transplant-ing and just prior to harvesting, using the acetylene reduc-tion assay, or ARA (Myrold et al. 1999). The containerswere placed in 1.5 L glass jars with their shoots protruding.The jars were sealed with reusable adhesive (Tac’N Stick,Ross Products), and 10% of the air volume was replacedwith acetylene. After a 1 h incubation at ca. 20 8C, gasfrom the jars was analyzed using a Varian 3400 GC fittedwith a 1 mL sampling valve and a Haysep T column. Threetypes of control jars were used for the ARA: (i) jars withoutplants with 10% acetylene were used to account for ethylenein the acetylene gas, (ii) jars with plants but no acetylenewere used to account for non-nitrogen fixation related produc-tion of ethylene, and (iii) jars with non-nodulated plants withacetylene were used to account for nonsymbiotic nitrogen fix-ation. These last controls were run at the end of the experi-ment, and plant roots were examined after the ARA to ensurethe plants did not have nodules. Only control type 1 jars hadany measurable ethylene. Plants were deemed to be fixing ni-trogen when more than 2.1 nmol�mL–1 ethylene was detected,that is, 2 standard deviations greater than the mean concentra-tion of ethylene in the acetylene used in the incubation.

Every other week, the height and basal diameter of theeach plant were measured. These data were then used to es-timate shoot dried mass of plants throughout the experimentas follows. A linear regression of the dried shoot mass to theheight multiplied by diameter squared of plants at the timeof harvest was performed. The slope and intercept of the lin-ear regression were then used to estimate shoot dried massat different times throughout the experiment from the bi-weekly height and diameter measurements. Relative growrates (RGRs) were estimated between weeks 2–6 and weeks7–11 after transplanting. This was done by dividing the dif-ference in the natural log-transformed estimates of shoot drymass by the number of days in the measurement period(Hunt 1978).

Plants were harvested 20 weeks after transplanting. Rootswere washed and placed in a 40 cm � 40 cm glass trayfilled with water to disperse the root. The roots were exam-ined for the presence of nitrogen-fixing nodules and ectomy-corrhizae. A glass plate was then placed on top of the rootsto minimize shadow effects, and the roots were scanned us-ing a flatbed scanner. Root length was then determined us-ing the macro by Kimura and Yamasaki (2001) in NIHImage version 1.63. From each plant, about 15 sections ofroots 5 cm long were examined microscopically. These sec-tions were first examined under a dissecting microscopethen cleared with 3 mol�L–1 KOH at 70 8C for 1 h, rinsedrepeatedly with water, acidified in 1% HCl, and stainedwith trypan blue (Johnson et al. 1999). Plants were scoredas being ectomycorrhizal by the presence of a mantle. Theremaining roots and shoots were dried at 65 8C and weighedfor biomass estimates. Dead plants were examined for thepresence of nodules, but their mass was not included as partof the plant growth analysis.

A number of plants were excluded from the final analysis.Twelve plants that were not inoculated but developed nod-ules were excluded. Seven plants, four that were not inocu-lated with P. involutus and three that were, had hyphae inthe cortical region of the roots but no mantle. These werealso excluded. A general problem in inoculation experimentswith symbiotic organisms is what to do in the case of inocu-lated plants that do not have detectable symbioses at thetime of harvest. Although an inoculated plant may not havea developed symbiosis at the time of harvest, the symbiontmay still have had an effect on its performance. Since notall inoculated plants in this experiment showed obvioussigns of colonization by the symbionts at the end of the ex-periment, the analysis includes two types of comparisons.The first includes all plants in each inoculation treatment.The second compares plants in each treatment that werecolonized versus those that were not. Colonization and mor-tality data were analyzed using contingency analysis andgrowth data using ANOVA and Tukey’s HSD post hoc testson significant effects. Plant mass data were log transformedto create homogeneous variances between treatment groups.To examine the effect of the treatments on root morphology,ANCOVA was applied to the root length data, using rootmass as a dependent variable. This allows root length datato be analyzed with reference to root mass, when root massvaries between treatments. Where interactions between thecovariates (inoculum and media effects) and the root masswere significant, Tukey’s HSD test was performed to com-pare multiple slopes assuming unequal variances (Zar1984). The statistical analyses were performed using JMPIN version 4.0.4.

Results

Overall plant mortality was 13.4%. Planting media had asignificant effect (w2 = 5.8) on plant mortality, with a rate of3.5% on the peat, 17.9% on the peat–tailings, and 15.0% onthe tailings media. Within each planting medium, the onlysignificant inoculation effect on mortality occurred on thepeat–tailings: inoculating plants with Frankia alone signifi-cantly increased mortality (Table 1). There was a significantdifference in mortality of those plants that did develop nod-

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ules versus those that did not (w2 = 14.2). None of the nodu-lated plants on the peat–tailings died, whereas those thatwere inoculated but did not develop nodules had a mortalityof 40%. On each medium, plants inoculated with bothFrankia and P. involutus always had the lowest mortalityrate of all the inoculation treatments.

Overall, 45.9% of the Frankia-inoculated plants devel-oped nodules. Of those plants that produced nodules, theaverage number of nodules per plant was 3.6 (±0.4 SE).The probability of a plant developing nodules and the num-ber of nodules per plant were significantly affected by theplanting media and whether or not plants were also inocu-lated with P. involutus. Frankia-inoculated plants that werealso inoculated with P. involutus were more likely to de-velop nodules (55.5%) than plants inoculated with Frankiaalone (33.3%, w2 = 5.4). Significantly more plants than ex-pected on the peat medium (90.0%, w2 = 13.3) and signifi-cantly fewer than expected plants on the tailings medium(11.9%, w2 = 10.6) developed nodules. Similarly, of thoseplants that developed nodules, plants grown on peat had sig-nificantly more nodules (4.6 ± 0.5) than plants grown onpeat–tailings (2.3 ± 0.7) or pure tailings (1.0 ± 1.3).

Overall, 23.1% of the plants inoculated with P. involutuswere classified as ectomycorrhizal at the end of the experi-ment. Although there was no effect of the Frankia inocula-tion on mycorrhizal formation, significantly more plants thatdeveloped nodules also developed ectomycorrhizae (95.0%)compared with plants that did not develop nodules (35.2%,w2 = 23.6). Significantly more plants than expected (66.7%)developed ectomycorrhizae when grown on the peat me-dium, and significantly fewer (2.6%) developed ectomycor-rhizae when grown on tailings.

At 6 weeks after the transplanting, only one plant out ofthe 20 tested showed any nitrogenase activity according tothe ARA. By week 12 post-transplanting, 14 of the 28 plantstested that had nodules at the time of harvest were fixing ni-trogen. At the time of harvest, all of 18 nodulated plantstested were fixing nitrogen.

Both the planting media and the inoculation treatmentshad a significant effect on RGRs in weeks 2–6 and weeks7–12 after transplanting, but there was no interaction be-tween these treatments (Table 2). During weeks 2–6, theRGRs of plants inoculated with P. involutus were signifi-cantly greater than those of plants not inoculated withP. involutus. By weeks 7–11, the RGRs of P. involutus ino-culated plants were no different from the RGRs of the con-trol plants. Those plants that developed mycorrhizae hadsignificantly greater RGRs than inoculated plants that didnot develop mycorrhizae both in weeks 2–6 (3.17 ± 0.43 vs.1.78 ± 0.25 mg�g–1�d–1) and in weeks 7–11 (9.34 ± 0.70 ver-

sus 5.63 ± 0.39 mg�g–1�d–1). During weeks 7–11, plants ino-culated with Frankia had significantly lower RGRs than allother inoculation treatments. However, plants that had nod-ules at the end of the experiment had significantly greaterRGRs in weeks 7–11 (6.19 ± 0.72 mg�g–1�d–1) comparedwith inoculated plants that did not develop nodules (3.18 ±0.55 mg�g–1�d–1). Plants grown on the tailings medium hadsignificantly lower RGRs than plants grown on the othermedia during weeks 2–6 and significantly lower RGRs thanthe plants grown on the peat medium during weeks 7–11.

A two-way ANOVA showed that the effect of the inocu-lum on shoot dry mass varied with the planting medium(Fig. 1). On peat, plants inoculated with both Frankia andP. involutus had significantly greater shoot mass than eithercontrol or Frankia-inoculated plants. Since only two of theFrankia nodulated plants failed to develop nodules, no com-parison was made between inoculated plants that developedsymbioses versus those that did not. On the peat–tailingsmedium, plants inoculated with both Frankia andP. involutus had significantly greater shoot mass than allother inoculation treatments. Of the Frankia-inoculatedplants, those plants that had nodules at the end of the experi-ment had greater shoot mass (0.430 ± 0.970 g) than plantsthat did not develop nodules (0.111 ± 0.105 g), but the dif-ference may not be significant (p = 0.054). There were nodifferences in shoot dry mass between the P. involutusplants that had mycorrhizae at the end of the experimentversus those that did not. Plants that were inoculated withboth Frankia and P. involutus and were colonized by bothsymbionts had significantly greater shoot mass (0.827 ±0.123 g) compared with those that were not colonized(0.181 ± 0.117 g). On the tailings medium, there were nodifferences in shoot dry mass between the inoculation treat-ments. Frankia-inoculated plants that developed nodules hada significantly greater dried shoot mass (0.875 ± 0.068 g)than plants that did not develop nodules (0.136 ± 0.019 g).Only one of the surviving P. involutus inoculated plantsgrown on tailings had mycorrhizae at the end of the experi-ments, so no comparisons were made between inoculatedplants that were colonized versus those that were not.

A two-way ANOVA showed a significant effect of plant-ing medium on root/shoot ratios but no effect of the inocu-lation treatment or interaction between the inoculationtreatment and planting media. Plants grown on peat had sig-nificantly lower (p = 0.0014) root/shoot ratios (0.54 ± 0.05)than plants grown on either peat–tailings (0.92 ± 0.06) ortailings (0.90 ± 0.04). The relationship between root lengthand root mass was dependent on the interaction betweenplanting media and inoculation treatment (Fig. 2). As theproportion of tailings in the planting medium increased,roots became thinner. This effect was partially offset by in-oculating plants with Frankia and (or) P. involutus. Conse-quently, control plants grown on peat–tailings hadsignificantly thinner roots than all inoculation treatmentsgrown on peat. Control plants grown on tailings had signifi-cantly thinner roots than any of the inoculated plants grownon peat–tailings.

Discussion

Combined inoculation of plants with the nitrogen-fixing

Table 1. Percent mortality of plants receiving different symbiontinocula on different planting media.

Control FrankiaPaxillusinvolutus

Frankia +P. involutus

Peat 5.0 0 8.3 0Peat–tailings 9.1 38.5* 14.3 5.0Tailings 11.8 27.2 13.4 5.6

*Signficantly different within the planting medium according to aPearson w2 test.

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bacteria and mycorrhizal fungi resulted in increased plantyield in the peat media, and especially the peat–tailings me-dia, suggesting a synergistic effect on plant performance.The experiment also suggests that the presence of one sym-biont has a positive effect on the performance of the other.Dual inoculation increased the likelihood of nodule develop-ment, and the presence of nodules increased the likelihoodthat a plant would have mycorrhizae. Chatarpaul et al.(1989) have shown that the combined inoculation of ecto-mycorrhizal fungi and Frankia increased alder growth morethan either symbiont alone. Dual inoculation of bothFrankia and vesicular–arbuscular mycorrhizal forming fungiwas found to increase Hippophae tibetana mass, mycorrhi-zal infection, and acetylene reduction rates (Tian et al.2002). This synergistic effect is likely due to the fact thatFrankia and mycorrhizae provide plants with nitrogen andphosphorus, respectively. They therefore remove two differ-ent types of nutrient limitation and create a three-way mutu-

alism. Similar results have been found in legume, rhizobiabacteria, and mycorrhizal fungi systems (Smith and Read(1997). Given the similarities in the physiology of symbioticnitrogen fixation in actinorhizal plants and legumes (Gual-

Table 2. The effect of planting medium and inoculation treatmenton mean relative growth rates (mg�g–1�d–1 ± SE) during weeks 2–6and weeks 7–11.

Weeks 2–6 Weeks 7–11

MediumPeat 1.97±0.34b 7.32±0.69bPeat–tailings 1.50±0.30b 5.62±0.45abTailings 1.02±0.18a 5.19±0.41a

InoculumControl 0.54±0.19a 5.80±0.72bFrankia 0.48±0.18a 4.30±0.49aPaxillus involutus 1.42±0.34b 5.74±0.80bFrankia + P. involutus 2.46±0.28b 6.92±0.41b

Note: Values followed by different letters within a date and treatmentare significantly different according to Tukey’s HSD test.

Fig. 1. Effect of planting medium and inoculation treatment onshoot dry mass. Within a planting medium, bars with the same let-ters are not significantly different. Data are means with standarderror bars.

0

500

1000

1500

2000

2500

3000

3500

Inoculum

C. b = 1710 r2 = 0.48 A

F. b = 2230 r2 = 0.91 A

P. b = 1910 r2 = 0.62 A

F+P. b = 1730 r2 = 0.59 A

500

1000

1500

2000

2500

3000

Inoculum

C. b = 5090 r2 = 0.88 BC

F. b = 2990 r2 = 0.94 AB

P. b = 2780 r2 = 0.69 AB

F+P. b = 2450 r2 = 0.69 AB

0

500

1000

1500

2000

2500

3000

0 0.2 0.4 0.6 0.8 1.0 1.2 1.4

Inoculum

C. b = 8960 r2 = 0.79 C

F. b = 5200 r2 = 0.78 BC

P. b = 3230 r2 = 0.68 ABC

F+P. b = 6090 r2 = 0.82 BC

(A)

(B)

(C)

Ro

ot

len

gth

(cm

)

Root mass (g)

Fig. 2. Effect of peat (A), peat–tailings (B), tailings (C), and in-oculation treatments (C, control; F, Frankia; P, Paxillus involutus;F+P, Frankia and P. involutus) on the relationship between rootlength and mass. Letters in bold following the coefficient of deter-mination (r2) of least squares fits indicate significantly differentslopes (b) across all combinations of planting media and inocula-tion treatments according to a Tukey’s HSD test at p = 0.05.

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tieri and Bisseling 2000), the synergistic effect in both sys-tems is probably due to a common scenario.

A number of studies have documented the beneficial ef-fect of ectomycorrhizae (reviewed by Jentschke and God-bold 2000; Schutzendubel and Polle 2000) andendomycorrhizae (reviewed by Leyval et al. 1997) on thegrowth of plants exposed to heavy metals or heavy-metal-contaminated mine tailings. While mycorrhizae have beenshown to increase plant performance, there may be an upperthreshold of heavy metal concentrations beyond which nobenefit occurs. For example, Jones and Hutchinson (1986)found a beneficial effect of a number of ectomycorrhizalfungi on the performance of birch seedlings grown at low(34 mmol�L–1) but not high (85 mmol�L–1) concentrations ofnickel. The mechanisms for the benefit mycorrhizae provideto plants exposed to heavy metal is at present not clear butmay include prevention of enzyme inactivation, preventionof oxidation leading to premature lignification in roots(Schutzendubel and Polle 2000), and reduction of nutrientstress (Jentschke and Godbold 2000). In this study, part ofthe effect of mycorrhizal fungi inoculation on plant perform-ance is likely due in part to increased occurrence of nodula-tion. The mechanisms for this increase are likely due to amore favourable physiological status of the plants’ allowingfor increased colonization rates. Since inoculation with ei-ther symbiont increased root thickness when plants weregrown in the presence of tailings, and the tailings were lowin N and P, they likely increased plant performance by in-creasing the N and P nutrition in the plants.

Both Frankia and P. involutus colonization was sup-pressed by the presence of tailings. Little is known aboutthe effect of heavy metals on Frankia. Although Frankia isknown to be free living in the soil, it is usually associatedwith the presence of host or nonhost plant species (Smo-lander et al. 1988; Pashcke and Dawson 1992; Markhamand Chanway 1996). Microscopic analysis has shown thatFrankia grows in the rhizosphere of plant roots (Ronnko etal. 1993). Given the fact that this site is generally free ofvascular plants, it is unlikely that the inoculation potentialof the tailings for either Frankia or ectomycorrhizae is everhigh. Also, heavy metals generally have a direct effect onmycorrhizal fungi, decreasing the inoculation potential ofcontaminated soils (Leyval et al. 1997).

Although the presence of nodules on plants was associ-ated with greater growth, the overall effect of inoculatingplants with Frankia seems to be complex. Frankia inocula-tion was associated with decreased survival on peat–tailingsand decreased shoot RGRs early in the experiment. Whilenitrogen-fixing nodules provide a benefit to plants, in theearly stages of their development, bacteria show no signs ofnitrogen fixation (Berry and Sunell 1990), suggesting thatthey are a carbon drain on the plant without providing abenefit of fixed nitrogen. This is the first study that I amaware of that has shown a whole plant level reduction inperformance of an actinorhizal plant in the early stages ofnodule formation, before nitrogen fixation has started. Sincelegume nodules go through similar anatomical changes(Gualtieri and Bisseling 2000), plants may also show a re-duced performance early in nodule development. The com-bined effect of nodule formation and presence of low-nutrient, heavy-metal-laden tailings in the planting media

may therefore explain the increased mortality of Frankia-in-oculated plants grown on peat–tailings. A benefit to theplant from inoculation with nitrogen-fixing bacteria mighttherefore be better realized by delaying transplanting untilnodules have fully developed. The fact that plants inoculatedwith the ectomycorrhizal fungi did not show an early reduc-tion of RGR suggests that there is less cost associated withthe development of ectomycorrhizae. It has been suggestedthat the ectomycorrhizae that develop on alders withP. involutus (Massicotte et al. 1999) and other fungi(Godbout and Fortin 1983) do not have a well-developedHartig net. Godbout and Fortin (1983) have suggested thisis a common occurrence in the angiosperms. It may be that,in more developed ectomycorrhizal associations, the earlydevelopment of the symbiosis is associated with decreasedplant performance. Since inoculating plants with the ecto-mycorrhizal fungi negated the negative effect of the Frankiainoculum on plant growth early in the experiment, it may bethat ectomycorrhizae can benefit plants much earlier in thedevelopment of the symbiosis than nitrogen-fixing bacteria.Overall these results suggest that during the early stages ofdevelopment, there is less of a cost associated with ectomy-corrhizae than nitrogen-fixing bacterial symbioses.

AcknowledgementsThis work was supported by a Natural Sciences and Engi-

neering Research Council of Canada operating grant and aCanadian Foundation for Innovation New Opportunities grantto the author and a NSERC Aboriginal Summer StudentAward to Ira Morrisseau. I thank Sylvie Renault for logisticsupport and Melissa Day, Laura Lazo, and three anonymousreviewers for making suggestions to the manuscript.

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