Variability of nitrogen-fixing Frankia on Alnusspecies

John H. Markham

Abstract: Plants maintain mutualistic symbioses with multiple symbiont genotypes that differ in the benefits they provide.To investigate differences in the effect of nitrogen-fixing Frankia on Alnus species, spore-producing (sp+) nodules fromAlnus rubra Bong. and Alnus incana subsp. rugosa (Du Roi) Clausen and non-spore-producing (sp–) nodules fromAlnus viridis subsp. crispa (Ait.) Turrill, A. rubra, and A. incana subsp. rugosa were collected from each of four differentpopulations and used to inoculate all three Alnus species. As expected, sp+ Frankia produced significantly more noduleson all three species. However, A. crispa, which normally does not have sp+ nodules in the field, was more susceptible to ahigh level of infection by sp+ Frankia in general, and by any source of sp+ Frankia in particular, whereas A. incanasubsp. rugosa, which has the highest abundance of sp+ in the field, was less susceptible to high levels of infection. Thissuggests that A. incana subsp. rugosa develops resistance to high levels of infection. The infectivity of an sp+ Frankiasource on A. viridis subsp. crispa and A. rubra was positively correlated with the proportion of sp+ nodules on the site itwas collected from, suggesting that the variation in the abundance of sp+ in the field is caused by sp+ Frankia with differ-ent levels of infectivity. There was no effect of Frankia sources on nodule allocation. Plant growth was positively corre-lated with the specific nodule mass and the specific nodule activity, and negatively correlated with the nodule number perplant. Sp+ Frankia resulted in significantly smaller plants in A. rubra. While there was no overall sp+ type effect on thegrowth of A. viridis subsp. crispa, the largest plants always resulted when they were inoculated with sp–, and the smallestwith sp+ Frankia. Neither spore type nor inoculum source had any effect on the performance of A. rugosa. These resultssuggest that Alnus species remain susceptible to infection by both Frankia spore types, but are able to modulate the effec-tiveness of these spore types when they are the common symbionts in the field.

Key words: symbiosis, infectivity, nodulation, nitrogen fixation, mutualism.

Resume : Les plantes maintiennent des symbioses mutualistes avec de multiples genotypes de symbiotes, leur procurantdes benefices varies. Afin d’examiner les variations des effets des Frankia fixateurs d’azote sur les especes d’Alnus,l’auteur a recolte des nodules producteurs de spores (sp+) chez l’Alnus rubra Bong. et l’Alnus incana subsp. rugosa (DuRoi) Clausen, et non producteurs de spores (sp–) chez l’Alnus viridis subsp. crispa (Ait.) Turrill, l’ A. rubra et l’ A. in-cana subsp. rugosa, a partir de chacune de quatre populations, et les a inoculees sur les trois especes d’Alnus. Comme ons’y attendait, les Frankia sp+ ont produit significativement plus de nodules chez les trois especes. Cependant, l’A. crispaqui normalement ne porte pas de nodules sp+, s’est montre plus susceptible a de fortes colonisations par les Frankia sp+,en general, et par toute source de Frankia sp+ en particulier, alors que l’A. incana subsp. rugosa, qui porte la plus forteabondance de sp+ sur le terrain, s’est montre moins susceptible a de fortes colonisations. Ceci suggere que l’A. incanasubsp. rugosa developpe une resistance a la colonisation. Le pouvoir colonisateur d’une source de Frankia sp+ sur l’A. vi-ridis subsp. crispa et l’A.rubra montre une correlation positive avec la proportion de nodules sp+ sur le terrain ou il a eterecolte, ce qui suggere que la variation de l’abondance des nodules sp+ aux champs, proviendrait de Frankia sp+ munisde divers pouvoirs colonisateurs. On n’observe aucun effet des sources de Frankia sur l’allocation des nodules. La crois-sance des plantes montre une correlation positive avec la masse nodulaire specifique et l’activite nodulaire specifique,alors que la correlation est negative avec le nombre de nodules par plant. Les Frankia sp+ entraınent la formation deplants plus petits chez l’A. rubra. Alors qu’il n’y a pas d’effet general du type sp+ sur la croissance de l’A. viridis subsp.crispa, les plantes les plus grandes proviennent toujours d’inoculation avec sp–, et les plus petites d’inoculations avec desFrankia sp+. Ni le type de spore ni la source d’inoculum n’affectent la performance d’A, rugosa. Les resultats suggerentque les especes d’Alnus demeurent susceptibles a la colonisation par les deux types de spores du Frankia, mais peuventmoduler l’efficacite de ces types spores lorsqu’elles constituent des symbiotes communs sur le terrain.

Mots-cles : symbiose, pouvoir colonisateur, nodulation, fixation de l’azote, mutualisme.

[Traduit par la Redaction]

Received 14 December 2007. Published on the NRC Research Press Web site at botany.nrc.ca on 18 April 2008.

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

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IntroductionSymbiotic relations between plants and soil microbes can

differ in the degree of benefit one or both of the partners re-ceive(s). Conditions thought to allow a relationship to be-come mutualistic include a low cost:benefit ratio,persistence (fidelity), partner choice, and the ability to sanc-tion ineffective partners (Denison 2000; Stadler and Dixon2005; Foster and Wenseleers 2006). Unlike parasitic interac-tions, which tend to be highly specific between taxa, mu-tualistic interactions tend to be less specific — typically asymbiont can form a relationship with many host species(Law and Lewis 1983; Bidartondo et al. 2002; Sanders2003). Since symbionts can differ in the benefit they pro-vide, there should be selection for plants that can recognize,reduce colonization by, or alter the effectiveness of, inher-ently less effective genotypes. We know for example thatcheater symbionts (those that provide no benefit) can colo-nize hosts (Hahn et al. 1990; Genkai-Kato and Yamamura1999) and hosts can develop resistance to them (Wolters etal. 1999). So, while hosts may show a low absolute specific-ity (i.e., they can form symbioses with many microbialtaxa), there may be host preferences between plants andsymbionts (Sanders 2003), and spatial structuring of the ef-fectiveness of plant symbioses can occur at regional scales(Lie et al. 1987; Chanway and Holl 1993), between sites(Parker 1995; Spoerke et al. 1996), or within sites (Chanwayet al. 1989; Bever et al. 1996).

Symbiotic nitrogen fixation with the actinomyceteFrankia occurs in nine plant families. There is not a strongcorrespondence between the phylogenies of the plant andbacterial taxa as a whole, with more primitive hosts tendingto be more promiscuous (Benson and Clawson 2000). How-ever, there is some degree of specificity at the plant familylevel (Torrey 1990) and the effectiveness of plant–Frankiacombinations can vary from one plant species to another(Dillon and Baker 1982; Teissier du Cros et al. 1984;Sellstedt et al. 1986; Mansour and Baker 1994) or betweenpopulations of the same plant species (Markham and Chan-way 1999). This suggests that some degree of coevolution(sensu Janzen 1980) or specialization has occurred betweenthe plant and bacterial taxa, with periodic shifts betweentaxa (Benson and Clawson 2000). Within some host-specificgroups of Frankia, including those that form nodules withAlnus species, two morphological groups have beendescribed — those that do and those that do not producespores within nodules (hereinafter referred to as sp+ andsp– Frankia, respectively). Both inoculation (van Dijk1978; VandenBosch and Torrey 1984, 1985) and moleculargenetic studies (Simonet et al. 1994) have shown that theability to produce spores in nodules is a genetic trait ofthe Frankia genotype. This ability may be an ecologicallyimportant trait, since sp+ Frankia have been shown to bemore infective (i.e., form more nodules for a given quan-tity of Frankia) than sp– Frankia (Houwers and Akker-mans 1981; van Dijk 1984; Wheeler et al. 1986). Sporeproduction can also be associated with reduced nitrogenaseactivity (Houwers and Akkermans 1981; VandenBosch andTorrey 1984; van Dijk 1984). It could therefore be arguedthat sp+ Frankia are less mutualistic (i.e., have more of acheating strategy) than sp– Frankia. If plants were capableof responding effectively to variation in Frankia infective-

ness, we would expect that hosts on which sp+ Frankia aremore common would show a lower successful infectionrate and (or) less of a decrease in effectiveness of theplant–Frankia combination than those hosts with sp– Frankia.

The purpose of this study was to examine the variationthat sp+ and sp– Frankia exhibit in the infection of and ben-efit to plants in a genus (e.g., Alnus) with which they arenaturally associated, albeit in varying abundance, dependingon the host species. Since within a location the genetic di-versity between sp+ (Simonet et al. 1994) and sp– Frankia(Clawson et al. 1999) can be low, with dominance by a sin-gle strain, a single source (sp+ or sp–) was selected fromeach of a number of sites. Since sp+ nodules cannot be iso-lated (Schwintzer 1990), crushed nodules were used as aFrankia source for both the sp– and sp+ Frankia.

Materials and methods

Nodules containing either sp+ or sp– Frankia were col-lected from three Alnus species: Alnus viridis subsp. crispa(Ait.) Turrill (hereinafter referred to as A. crispa),Alnus rubra Bong., and Alnus incana subsp. rugosa (DuRoi) Clausen (hereinafter referred to as A. rugosa). TheA. rubra nodules were collected from the west coast of Brit-ish Columbia and nodules from A. crispa and A. rugosafrom southeast Manitoba. Forty-two A. rubra sites and 12each of A. crispa and A. rugosa sites were sampled. In eachsite at least one nodule was collected from at least 20 differ-ent plants. Plants were usually selected by running a transectalong the long axis of the site and selecting the nearestplants to random points, at least 10 m from other points.Nodules were collected from within 1 m of the base of theplant to a depth of 10 cm. Nodules were stored on ice andthen held at 4 8C in the lab until they were examined micro-scopically. Hand-cut sections of nodule lobes were stainedin Fabil’s reagent (Noel 1964) and examined at 400� undera light microscope for the presence of spores (Markham andChanway 1998). If spores were not found in the first lobe, atleast one other lobe was examined. It was found that 83% ofthe A. rugosa, none of the A. crispa, and 43% of theA. rubra sites had at least one sp+ nodule. For long-termstorage, the nodules were surface-sterilized for 5 min in50% commercial bleach, rinsed repeatedly in sterile water,freeze-dried, and kept at –80 8C. From the 66 sampled sites,four sp– nodules (one per site) were randomly selected fromeach of the three Alnus species for an inoculation study(Table 1). Furthermore, four sp+ nodules were randomly se-lected from the A. rubra and A. rugosa stands. In addition tothese 20 Frankia sources, three sites were chosen where fournodules of the same spore type were compared in a parallelinoculation study.

Since nodules can contain more than one Frankia strain indifferent lobes (Reddell and Bowen 1985), a single nodulelobe was used as an inoculum source to reduce the possibil-ity of having more than one Frankia strain. It was assumedthat adjoining lobes on the same nodule (i.e., lobes clearlyarising from a bifurcation in the nodule and not directlyfrom the root) were of the same spore type as the lobes ex-amined microscopically. The mean dry mass equivalent ofthe selected lobes used to make the inocula was 0.015 ±0.001 g (mean ± SE). Each inoculum was prepared by

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crushing the lobe in a sterilized mortar and pestle in dilutesterile TE–PVPP buffer (0.1 mol�L–1 Tris HCl, 0.01MNa2EDTA with a suspension of 1 g�L–1 PVPP). The coarseplant material was allowed to settle, and the suspensiondiluted to the equivalent of 0.34 mg dry mass per mL. Eachsuspension was stored in three separated aliquotsat –80 8C. Seeds of each Alnus species were collectedfrom within the range of where the nodules were collected,but not from any of the sites used as a source of inocula.This was done to ensure that each inoculum applied toplants would come from a site from which it had not beencollected. Seeds were surface-sterilized with 95% ethanolfor one minute, rinsed, and germinated on sterile Turfacein a glasshouse with supplemental metal halide lighting(ca. 200 W�m–2 at bench level) for 16 h per day. Oncetrue leaves had formed, the seedlings were transplanted to3.8 cm � 14 cm deep planting tubes (Ray Leach Cone-tainers; Stuewe and Sons, Corvallis, Oreg.) containing80% Turface – 20% vermiculite (v/v). For 4 weeks, plantswere fertilized weekly with a full basic Rorison’s nutrientsolution (4 mmol�L–1 N, 1 mmol�L–1 P, 2 mmol�L–1 K,2 mmol�L–1 Ca, 1 mmol�L–1 Mg, 53 mmol�L–1 Fe, 9 mmol�L–1

Mn, 5 mmol�L–1 B, 1 mmol�L–1 Mo, 2 mmol�L–1 Zn, 2 mmol�L–1

Cu, Booth et al. 1993). Five weeks after transplanting, potswere repeatedly watered to leach out the nutrients. For theremainder of the experiment, the fertilizer was switched toa N-free formula with the same elements as before. Plantswere inoculated with a crushed nodule source 6 weeksafter transplanting by injecting 1 mL of the crushed nodule

suspension into the rooting zone of the plants. This amountof crushed nodules is far below the levels used for inocu-lation in other studies (e.g., 16.7 mg�mL–1 fresh mass inMarkham and Chanway (1998) and Huss-Danell (1991). Apreliminary experiment showed that this amount of crushednodules does not restrict nodule formation for even highlyinfective Frankia, whereas when higher quantities are used,plants reach a plateau in nodule numbers. For each of the20 inocula, 16 plants of each Alnus species were inocu-lated (320 plants of each of the three Alnus species).Although contamination from ambient Frankia is generallynot a problem with this type of assay, each inoculatedplant was surrounded by noninoculated control plants inthe planting trays to monitor Frankia contamination. Owingto space limitations and to the time required to conductacetylene reduction measurements, the experiment was div-ided into three growth trials: all Alnus species and sourcesof inoculum were used in each of the trials with a mini-mum of four replicates for each inoculum–species combi-nation. Plants were harvested 12 weeks after beinginoculated. Infectivity was measured as the mean numberof nodules per plant and the infection rate per inoculum,i.e., the percentage of inoculated plants that produced nod-ules. The effectiveness of each inoculum in fixing nitrogenwas measured as the total plant dry mass and the nitrogenfixation rate of the nodulated plants. Since alders havesmall seeds, and plants were raised on a nitrogen-free me-dium for most of the experiment, total biomass should be agood indicator of total nitrogen fixed (Mytton 1984), as

Table 1. Location of nodules (latitude and longitude) used for inoculation testsand proportion of spore positive (percent sp.+) nodules found at each site.

Nodule spore type

Site code N W Sp– Sp+ Sp+ (%)

A. crispa sitesAs1 50839.471’ 95824.062’ c–1 0Ac1 49836.133’ 96806.734’ c–2* 0Ac11 49836.788’ 96807.123’ c–3 0Ac2 50839.483’ 95818.575’ c–4 0

A. rubra sitesPTA 50827’ 127828’ b–1 0Sarita 48853’ 124852’ b–2 b+1 43GBC 49816’ 122835’ b–3 11MTM 50816’ 125830’ b–4 42SW16 49815’ 123814’ b+2 59Nit 48850’ 124839’ b+3 11PMDR 49830’ 123833’ b+4 18

A. rugosa sitesWSPP 49845.002’ 95831. 500’ g–1 g+1* 5Ar2 50804.048’ 95833.727’ g–2* g+2 55As1 50808.016’ 96801.518’ g–3 35Ar22 50806.050’ 96835.783’ g–4 50Ar3 49849.206’ 95815.588’ g+3* 30Ar1 50808.015’ 96801.516’ g+4 20

Note: Inocula are coded by host species (c, Alnus crispa; b, Alnus rubra; g, Alnus ru-gosa) and spore status (+ or –). All A. crispa sites were upland Pinus banksiana Lambdominated stands; all A. rubra sites were established on conifer harvested sites; and all A.rugosa sites were streamside habitats. The asterisks indicate sources where more than oneinoculum were compared within a site.

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well as growth. However, because differences in nitrogencontent could be due to differences in inorganic nitrogenabsorption during the seedling establishment phase, nitro-gen fixation was also assessed at the time of harvest. Theefficiency of nitrogen fixation was measured as specific nod-ule activity (SNA), i.e., the amount of nitrogen fixed per nod-

ule dry mass. Nitrogen fixation was measured using theacetylene reduction assay (Myrold et al. 1999) and soSNA was expressed as mmol C2H4�h–1�mg–1 nodule mass.All measurements were made between 1000 and 1400 hon the day a plant was harvested to avoid any diurnaldrop in acetylene reduction activity (Tripp et al. 1979).

Fig. 1. Nodulation of Alnus viridis subsp. crispa, Alnus rubra, and Alnus incana subsp. rugosa by inocula. Filled bars, sp– inocula; openbars, sp+ inocula. Values above bars are the percentage of plants that formed nodules. Within plant species, inocula with the same letterabove the bar are not significantly different according to Tukey’s HSD test (p = 0.05). Inocula with less than 19% of the plants nodulatedwere excluded from the analyses and therefore do not have letters. Bars are means ± 1 SE.

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Planting medium was washed from the roots and the wholeexcised root system was then incubated for one hour in anatmosphere with 10% acetylene. Gas samples were ana-lyzed in a Varian 3400 GC with a 1 mL sampling valveand a Haysep T column (Varian Canada Inc., Edmonton,Alta.). For every five samples, three types of controlswere analyzed. The first consisted of only 10% acetyleneand was used to account for ethylene in the acetylene gas.The second consisted of a non-nodulated root system froma control plant with 10% acetylene to account for any non-

symbiotic nitrogen fixation. The third control was a plantroot without any acetylene, used to account for ethyleneproduction not related to nitrogen fixation. Only the firstcontrol type produced any measurable ethylene, thereforethe proportion of ethylene in the acetylene was subtractedfrom the treatment bottles. For statistical analyses, only in-ocula that produced three or more nodulated plants werecompared when examining differences between inocula.Harvested nodules were kept refrigerated and sub-samplesof nodules from each plant were examined microscopicallyfor spores. None of the plants inoculated with sp– inoculahad spores. Of the plants inoculated with sp+ inocula, allhad nodules with spores, but some of the small noduleshad not yet developed sporangia at the time of harvest.The remaining nodules were freeze dried and the total nod-ule dry mass estimated. The specific nodule mass (SNM)was calculated as the average mass per nodule for eachplant (i.e., the total nodule mass / nodule number). Noduleallocation was calculated as nodule dry mass, as the per-cent of total plant dry mass and was considered as a meas-urement of the plant investment in the symbiosis.

To determine whether there were differences between sp+and sp– Frankia, and to avoid pseudo-replication, mean val-ues per inoculum were compared using one-way ANOVAsfor each test species. To compare different inocula from thesame site, separate analyses were performed. Only threecontrol plants, all in the second growth trial and located ad-jacent to one another on the greenhouse bench, developednodules. Excluding the surrounding experimental plantsfrom the analysis had no effect on the overall trends. Thedata were analyzed using a GLM with JMP version 5.1.

ResultsThe mean number of nodules per plant varied signifi-

cantly among the different inocula, ranging from 0 to 96

Table 2. Effect of Frankia spore type on plant performance.

Spore type

Positive Negative P valuePlant mass* (mg) A. crispa 401.±32 475.±38 0.163

A. rubra 147.±36 335.±62 0.039A. rugosa 370.±28 450.±60 0.317

Nodule no. A. crispa 56.1±13.1 0.26±0.02 <0.001A. rubra 21.2±9.3 0.21±0.12 0.011A. rugosa 8.6±3.7 1.2±0.2 0.023

Allocation (%) A. crispa 2.7±0.2 2.6±0.2 0.619A. rubra 5.1±0.9 6.0±1.0 0.542A. rugosa 4.8±0.3 4.2±0.3 0.158

SNM* (mg) A. crispa 1.6±1.5 10.9±1.3 <0.001A. rubra 1.2±0.6 19.2±8.3 0.051A. rugosa 5.9±1.6 10.8±1.3 0.029

SNA (mmol C2H4�h–1�mg–1) A. crispa 32.2±7.2 35.9±9.1 0.665A. rubra 2.7±2.2 6.6±3.2 0.338A. rugosa 17.1±5.1 24.0±4.6 0.383

C2H4�plant mass–1 (nmol�mg–1�h–1) A. crispa 827.±127 788.±158 0.868A. rubra 206.±187 466.±202 0.368A. rugosa 721.±147 798.±131 0.701

Note: Values are means ± SE for each inoculum tested. P values are the alpha values of ANOVA tests comparing the means of inoculaof each spore type. The asterisks indicate ANOVAs performed using log transformed data. SNM, specific nodule mass; SNA, specificnodule activity.

Fig. 2. The relationship between the infectivity of a sp+ Frankiasource on different Alnus species and the abundance of sp+ noduleson the site the source was collected from. Lines are least squaresfits.

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nodules per plant (Fig. 1). Only sp+ inocula were highly in-fective (producing more than 10 nodules per plant). Frankiafrom sp+ inocula produced significantly more nodules on allthree Alnus species (Table 2). However, the differences be-tween sp+ and sp– inocula were much more pronounced onA. crispa (216 times more nodules per plant with sp+ inocula)and A. rubra (101 times) than A. rugosa (8 times). Also,more of the sp+ inocula produced significantly more nod-ules than any of the sp– inocula on A. crispa (six out of

the eight inocula) and A. rubra (four out of eight) than onA. rugosa (two out of eight). Any inocula that were nothighly infective on A. crispa were also not highly infectiveon A. rubra or A. rugosa. Similarly, any inocula that werenot highly infective on A. rubra were not highly infectiveon A. rugosa. Any inocula that were on average highly in-fective had an infection rate of 100%, i.e., they nodulatedall 16 replicate plants. For all species, rates of infectionwere significantly higher for sp+ Frankia (80 ± 13%,

Fig. 3. The effect of different Frankia sources on plant dry mass.

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70 ± 10% and 83 ± 8% on A. crispa, A. rubra andA. rugosa, respectively) than for sp– Frankia (23 ± 17%,11 ± 8% and 56 ± 7%), with differences being less pro-nounced on A. rugosa.

For each species, nodule allocation did not differ betweeninocula or between inocula spore type. However, A. crispaplants had significantly lower nodule allocation than eitherA. rubra or A. rugosa regardless of the inocula (Table 2).The greater number of sp+ nodules per plant, combinedwith the lack of difference in allocation, meant that theSNM resulting from inoculation with sp– Frankia wassignificantly greater than that resulting from sp+ Frankia inall three species, with the greatest difference occurring inA. rubra. There was also a significant negative linear rela-tionship between the mean log SNM of an inoculum andthe number of nodules per plant across all three species(r2 = 0.751, p < 0.0001). The host species from which thenodules were collected had no effect on the number ofnodules formed on any of the tested species. There was apositive relationship between the proportion of sp+ nodulesfound on the site the inoculum was collected from and theinfectivity of that sp+ inoculum on A. crispa (r2 = 0.602,p = 0.023) and A. rubra (r2 = 0.517, p = 0.044), but noton A. rugosa (r2 = 0.309, p = 0.152, Fig. 2).

The mass of nodulated plants of A. crispa and A. rubra,but not A. rugosa, varied significantly between the differentinocula (Fig. 3). Alnus crispa plants inoculated with one ofthe sp– inocula (c–2) were significantly larger than plantsinoculated with sp+ inocula b+1 or b+4. As well, A. rubraplants inoculated with sp– inocula c–4 or g–1 were signifi-cantly larger than plants inoculated with either of three sp+inocula: b+1, g+2, or g+3. Owing to the variability betweeninocula of each spore type, sp+ inocula resulted in signifi-cantly lower plant mass than sp– inocula on A. rubra only(Table 2). SNA was significantly lower in A. rubra than ineither A. crispa or A. rugosa. Alnus rubra was also the onlyspecies that showed significant differences in SNA betweenthe inoculation sources. The two inocula producing signifi-cantly larger A. rubra plants (c–4 and g–1) had significantly

higher SNA (10.7 ± 3.4 and 15.6 ± 5.7 mmol�mg–1�h–1,respectively) than two of the four inocula that resulted insignificantly smaller plants (g+2, SNA = 0.7 ± 0.5, andg+3, SNA = 1.2 ± 0.6 mmol�mg–1�h–1). For all three species,the variation in plant mass between the inocula was best ex-plained by variation in the SNM (Fig. 4). The variation inplant mass could also be explained by the variations inSNA and ethylene reduced per plant mass (r2 = 0.251), butnot the variation in nodule allocation (r2 = 0.095) or numberper plant (r2 = 0.002). ANCOVA indicated that plant speciesand the inoculum spore type did not affect these relation-ships.

When Frankia of the same spore type within sites werecompared, there were few differences in infectivity or effec-tiveness in nitrogen fixation. With the inocula from siteWSPP, one of the sp+ inocula sources produced signifi-cantly fewer nodules per A. rugosa plant (0.5 ± 0.3) thanthe other inocula (combined average of 1.7 ± 0.4). Anotherinoculum produced a significantly lower number of noduleson A. crispa (0.5 ± 0.3) than the other three inocula (com-bined average of 2.7 ± 1.6). Overall these differences aresmall compared with the high rates of nodule formationfound in other sp+ inocula, suggesting low functional ge-netic diversity within sites.

Discussion

It has been argued that both the specificity (Hoeksema1999) and the effectiveness (Markham and Chanway 1999)of a symbiosis should increase with the chances of findingand maintaining a relationship with a symbiotic partner.This depends on either the partners being present, or beingable to survive until they are established in a symbiotic rela-tionship. For reasons not understood, sp+ are less likely thansp– Frankia to be found free living in the soil (van Dijk etal. 1988), resulting in sp– Frankia being more common insoils that lack host plants. If sp+ Frankia cannot be assuredof having continued access to a host, then theoretically, theywould be likely to trade off nitrogen fixation effectiveness

Fig. 4. The relationship between plant dry mass and (a) mean mass per nodule, and (b) SNA. Each point is the mean of each speciesinoculated with each of the different sp+ (open symbols) or sp– (filled symbols) inocula.

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on hosts for a short-term increase in their own fitness. Thealder species used in this study likely vary in how persistentthey are at a site, and therefore, how reliable they are as po-tential hosts. Alnus crispa is typically found in the under-storey of upland conifer sites, which are prone to standreplacement fires in the study region. Casual observationsindicated that A. crispa survives the typical spring fires thatdo not burn the forest floor, is persistent through conifercrown closure, and can spread vegetatively. By contrast,A. rubra is generally considered an early successional spe-cies being replaced by nonhost conifers (Harrington 1990).Markham and Chanway (1999) concluded that the lack ofpersistence of A. rubra on a site could explain the reducedperformance of its seedlings in field sites inoculated withFrankia from their parent populations. Alnus rugosa is usu-ally found in wetlands or along small streams typical tothose sampled in this study; however, it does not tolerateflooding (Kaelke and Dawson 2003, but see also Holmanand Schwintzer 1987) and so its persistence in these sites ishard to assess. To further comparative work between actino-rhizal species, we will need more data on the basic ecologyof the host species.

The results obtained here show that sp+ Frankia can onlybe considered more infective and less effective at nitrogenfixation on hosts, such as A. crispa and A. rubra, with whichthey are not commonly associated. Although sp+ inocula asa group produced significantly more nodules than sp– inoc-ula on all three species, the differences were much less pro-nounced on A. rugosa; on this species, there were nodifferences in plant performance between any of the inocula.This suggests that alders that are commonly in contact withsp+ Frankia may develop resistance to high infection rates.The pattern fits with general models of virulence that predictthat for any population resistance increases with exposure(Roy and Kirchner 2000). Alnus crispa was the most suscep-tible to high infection rates by sp+ Frankia and had the low-est abundance of sp+ Frankia in the field, both in the sitessampled in this study and in the survey of southern Quebecby Normand and Lalonde (1982). Since sp+ Frankia canform nodules on A. crispa, the lack of sp+ nodules in thefield may be due to some environmental factors limiting thepresence of sp+ Frankia on sites occupied by A. crispa. Thesituation among European Alnus where both Frankia sporetypes are also present seems more extreme. Sp+ Frankiaare normally found on A. incana, where they form effectivenodules that have the same effect on plant growth as sp–Frankia (Kurdali et al. 1990). However, inoculating Alnusglutinosa (L.) Gaertn., which normally have sp– nodules,with sp+ Frankia from A. incana results in totally ineffec-tive (i.e., non-nitrogen-fixing) nodules (Weber et al. 1987;van Dijk et al. 1988; Kurdali et al. 1989).

Differences in plant growth between the inoculation treat-ments could not always be explained by variation in noduleSNA. Plant growth was best predicted by nodule size(SNM), which itself was a function of nodule number. Itwould seem then that Alnus maximizes growth by having asmall number of large nodules. While some studies haveshown decreased nitrogenase activity in sp+ nodules (Normandand Lalonde 1982; Simon et al. 1985; Wheeler et al. 1986;Kurdali et al. 1990), this does not seem to be a universaltrend (Hall et al. 1979; Schwintzer 1990). Even if SNA is

the same between different nodules, the amount of nitrogenfixed per respired carbon may be lower in sp+ nodules(Monz and Schwintzer 1989). Since Alnus nodules canmake up to half of the total respiration of roots (Lundquist2005), this may be a substantial proportion of respired car-bon. Nodules may, therefore, have the same efficiency interms of mass allocated to them, but be less efficient onthe basis of their activity. Studies, both with actinorhizalplants and legumes, have shown that plant growth maychange with different host or symbiont genotypes becauseof a number of mechanisms including SNA (Dillon andBaker 1982; Zhang et al. 1997) or other measures of thesymbiotic interaction, such as the total proportion of plantnitrogen-derived from fixation (Mansour and Baker 1994),nodule-associated H2 production (Sellstedt et al. 1986;Huss-Danell 1991), or other unidentified processes (Carpenteret al. 1984). Studies on mycorrhizal symbioses have alsoshown that measures such as the proportion of roots colon-ized are not always good predictors of plant performance(Kernaghan 2005). Moreover, plants are not necessarilyfound with the most effective symbionts. For example,Dawson and Sun (1981) found that tested Alnus speciesexperienced superior growth with Frankia isolated fromComptonia peregrina (L.) J.M. Coult. than with Frankiafrom A. crispa. The data from an inoculation study of fourCasuarina species with four Frankia isolates by Mansourand Baker (1994) show a strong relationship between plantgrowth and the proportion of nitrogen in plant tissue thatwas fixed by the bacteria. However, one species showedits best performance with bacteria from a conspecific host,whereas another showed the poorest performance. It hasalso been shown that there are large populations of le-gumes infected by ineffective Rhizobium (Hagedorn 1978;Burdon et al. 1999). Screening of Australian Acacia spe-cies has also shown that most variation in Rhizobium effec-tiveness occurs between populations rather than betweenspecies, and that specificity is only common in rare Acaciaspecies (Thrall et al. 2000). Mytton (1984) has suggestedthat the effect of nitrogen-fixing bacteria on the perform-ance of legumes is highly variable. However, they are notnaturally nodulated with, or have the ability to be preferen-tially nodulated by, the most effective bacteria. I suggestsymbiotic nitrogen fixing organisms face a number of chal-lenges related to host–symbiont interactions. These includethe host needing to maintain the ability to be infected bymultiple symbiont genotypes; the predictability of host andsymbiont partner availability; the selection for symbionts tomaximize their own fitness; and the ability of hosts tomoderate the effectiveness of symbionts. This results in asituation where it is difficult to predict the effectiveness ofany particular host–symbiont combination.

AcknowledgementsThis work was supported by an Natural Sciences and En-

gineering Research Council of Canada Discovery Grant tothe author.

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