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 dexaminer les variations des effets des Frankia fixateurs dazote sur les especes dAlnus,lauteur a recolte des nodules producteurs de spores (sp+) chez lAlnus rubra Bong. et lAlnus incana subsp. rugosa (DuRoi) Clausen, et non producteurs de spores (sp) chez lAlnus 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 dAlnus. Comme onsy attendait, les Frankia sp+ ont produit significativement plus de nodules chez les trois especes. Cependant, lA. crispaqui normalement ne porte pas de nodules sp+, sest montre plus susceptible a de fortes colonisations par les Frankia sp+,en general, et par toute source de Frankia sp+ en particulier, alors que lA. incana subsp. rugosa, qui porte la plus forteabondance de sp+ sur le terrain, sest montre moins susceptible a de fortes colonisations. Ceci suggere que lA. incanasubsp. rugosa developpe une resistance a la colonisation. Le pouvoir colonisateur dune source de Frankia sp+ sur lA. vi-ridis subsp. crispa et lA.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 labondance des nodules sp+ aux champs, proviendrait de Frankia sp+ munisde divers pouvoirs colonisateurs. On nobserve aucun effet des sources de Frankia sur lallocation des nodules. La crois-sance des plantes montre une correlation positive avec la masse nodulaire specifique et lactivite nodulaire specifique,alors que la correlation est negative avec le nombre de nodules par plant. Les Frankia sp+ entranent la formation deplants plus petits chez lA. rubra. Alors quil ny a pas deffet general du type sp+ sur la croissance de lA. viridis subsp.crispa, les plantes les plus grandes proviennent toujours dinoculation avec sp, et les plus petites dinoculations avec desFrankia sp+. Ni le type de spore ni la source dinoculum naffectent la performance dA, rugosa. Les resultats suggerentque les especes dAlnus demeurent susceptibles a la colonisation par les deux types de spores du Frankia, mais peuventmoduler lefficacite de ces types spores lorsquelles constituent des symbiotes communs sur le terrain.
Mots-cles : symbiose, pouvoir colonisateur, nodulation, fixation de lazote, 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:email@example.com).
Botany 86: 501510 (2008) doi:10.1139/B08-023 # 2008 NRC Canada
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 plantFrankiacombinations 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 theplantFrankia 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 Fabils 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 TEPVPP buffer (0.1 molL1 Tris HCl, 0.01MNa2EDTA with a suspension of 1 gL1 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 Wm2 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 Rorisons nutrientsolution (4 mmolL1 N, 1 mmolL1 P, 2 mmolL1 K,2 mmolL1 Ca, 1 mmolL1 Mg, 53 mmolL1 Fe, 9 mmolL1Mn, 5 mmolL1 B, 1 mmolL1 Mo, 2 mmolL1 Zn, 2 mmolL1Cu, 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 mgmL1 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 inoculumspecies 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 fi...