9
PLANT MICROBE INTERACTIONS Frankia Populations in Soil and Root Nodules of Sympatrically Grown Alnus Taxa Anita Pokharel & Babur S. Mirza & Jeffrey O. Dawson & Dittmar Hahn Received: 25 April 2010 / Accepted: 13 July 2010 / Published online: 14 September 2010 # Springer Science+Business Media, LLC 2010 Abstract The genetic diversity of Frankia populations in soil and in root nodules of sympatrically grown Alnus taxa was evaluated by rep-polymerase chain reaction (PCR) and nifH gene sequence analyses. Rep-PCR analyses of uncultured Frankia populations in root nodules of 12 Alnus taxa (n =10 nodules each) growing sympatrically in the Morton Arboretum near Chicago revealed identical patterns for nodules from each Alnus taxon, including replicate trees of the same host taxon, and low diversity overall with only three profiles retrieved. One profile was retrieved from all nodules of nine taxa (Alnus incana subsp. incana, Alnus japonica, Alnus glutinosa, Alnus incana subsp. tenuifolia, Alnus incana subsp. rugosa, Alnus rhombifolia, Alnus mandshurica, Alnus maritima, and Alnus serrulata), the second was found in all nodules of two plant taxa (A. incana subsp. hirsuta and A. glutinosa var. pyramidalis), and the third was unique for all Frankia populations in nodules of A. incana subsp. rugosa var . americana. Comparative sequence analyses of nifH gene fragments in nodules representing these three profiles assigned these frankiae to different subgroups within the Alnus host infection group. None of these sequences, however, represented frankiae detectable in soil as determined by sequence analysis of 73 clones from a Frankia-specific nifH gene clone library. Additional analyses of nodule populations from selected alders growing on different soils demonstrated the presence of different Frankia populations in nodules for each soil, with populations showing identical sequences in nodules from the same soil, but differences between plant taxa. These results suggest that soil environ- mental conditions and host plant genotype both have a role in the selection of Frankia strains by a host plant for root nodule formation, and that this selection is not merely a function of the abundance of a Frankia strain in soil. Introduction Members of the genus Frankia are generally described as nitrogen-fixing bacteria that form root nodules in symbiosis with more than 200 species of non-leguminous woody plants representing 25 genera of angiosperms [13]. Root nodule formation occurs in host infection groups, with frankiae belonging to the Elaeagnus and the Alnus host infection groups being the most studied [4]. Frankiae of the Elaeagnus host infection group form nodules on plants of the genera Elaeagnus, Hippophaë, Shepherdia, Myrica, Morella, and Colletia, and those of the Alnus host infection group on plants of the genera Alnus, Morella, and Comptonia, with some specifically nodulating plants of the genera Casuarina and Allocasuarina [5, 6]. While specific interactions between Frankia strains and individual plant species representing their respective host infection groups are well documented [4, 7, 8], information on intrageneric variation in host plant compatibility with specific Frankia populations is limited [9, 10]. A. Pokharel : B. S. Mirza : D. Hahn (*) Department of Biology, Texas State University, 601 University Drive, San Marcos, TX 78666, USA e-mail: [email protected] J. O. Dawson Department of Natural Resources and Environmental Sciences, University of Illinois at Urbana-Champaign, 1201 South Dorner Drive, Urbana, IL 61801, USA J. O. Dawson Morton Arboretum, Lisle, IL 60532, USA Microb Ecol (2011) 61:92100 DOI 10.1007/s00248-010-9726-2

Frankia Populations in Soil and Root Nodules of Sympatrically Grown Alnus Taxa

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Page 1: Frankia Populations in Soil and Root Nodules of Sympatrically Grown Alnus Taxa

PLANT MICROBE INTERACTIONS

Frankia Populations in Soil and Root Nodulesof Sympatrically Grown Alnus Taxa

Anita Pokharel & Babur S. Mirza & Jeffrey O. Dawson &

Dittmar Hahn

Received: 25 April 2010 /Accepted: 13 July 2010 /Published online: 14 September 2010# Springer Science+Business Media, LLC 2010

Abstract The genetic diversity of Frankia populations insoil and in root nodules of sympatrically grown Alnus taxawas evaluated by rep-polymerase chain reaction (PCR) andnifH gene sequence analyses. Rep-PCR analyses ofuncultured Frankia populations in root nodules of 12 Alnustaxa (n=10 nodules each) growing sympatrically in theMorton Arboretum near Chicago revealed identical patternsfor nodules from each Alnus taxon, including replicate treesof the same host taxon, and low diversity overall with onlythree profiles retrieved. One profile was retrieved from allnodules of nine taxa (Alnus incana subsp. incana, Alnusjaponica, Alnus glutinosa, Alnus incana subsp. tenuifolia,Alnus incana subsp. rugosa, Alnus rhombifolia, Alnusmandshurica, Alnus maritima, and Alnus serrulata), thesecond was found in all nodules of two plant taxa (A.incana subsp. hirsuta and A. glutinosa var. pyramidalis),and the third was unique for all Frankia populations innodules of A. incana subsp. rugosa var. americana.Comparative sequence analyses of nifH gene fragments innodules representing these three profiles assigned thesefrankiae to different subgroups within the Alnus host

infection group. None of these sequences, however,represented frankiae detectable in soil as determined bysequence analysis of 73 clones from a Frankia-specificnifH gene clone library. Additional analyses of nodulepopulations from selected alders growing on different soilsdemonstrated the presence of different Frankia populationsin nodules for each soil, with populations showing identicalsequences in nodules from the same soil, but differencesbetween plant taxa. These results suggest that soil environ-mental conditions and host plant genotype both have a rolein the selection of Frankia strains by a host plant for rootnodule formation, and that this selection is not merely afunction of the abundance of a Frankia strain in soil.

Introduction

Members of the genus Frankia are generally described asnitrogen-fixing bacteria that form root nodules in symbiosiswith more than 200 species of non-leguminous woodyplants representing 25 genera of angiosperms [1–3]. Rootnodule formation occurs in host infection groups, withfrankiae belonging to the Elaeagnus and the Alnus hostinfection groups being the most studied [4]. Frankiae of theElaeagnus host infection group form nodules on plants ofthe genera Elaeagnus, Hippophaë, Shepherdia, Myrica,Morella, and Colletia, and those of the Alnus host infectiongroup on plants of the genera Alnus, Morella, andComptonia, with some specifically nodulating plants ofthe genera Casuarina and Allocasuarina [5, 6]. Whilespecific interactions between Frankia strains and individualplant species representing their respective host infectiongroups are well documented [4, 7, 8], information onintrageneric variation in host plant compatibility withspecific Frankia populations is limited [9, 10].

A. Pokharel : B. S. Mirza :D. Hahn (*)Department of Biology, Texas State University,601 University Drive,San Marcos, TX 78666, USAe-mail: [email protected]

J. O. DawsonDepartment of Natural Resources and Environmental Sciences,University of Illinois at Urbana-Champaign,1201 South Dorner Drive,Urbana, IL 61801, USA

J. O. DawsonMorton Arboretum,Lisle, IL 60532, USA

Microb Ecol (2011) 61:92–100DOI 10.1007/s00248-010-9726-2

Page 2: Frankia Populations in Soil and Root Nodules of Sympatrically Grown Alnus Taxa

For members of the Alnus host infection group, forexample, differences in abundance of nodules were foundas a function of host plant species with nodule numbersconsistently being greatest on Alnus rubra, less on Alnusincana subsp. incana, and least on Alnus glutinosa [9].Similar studies, however, documented contradicting resultswith nodule numbers being higher on A. glutinosa than onA. incana subsp. incana [10]. Diversity of frankiae of theElaeagnus host infection group was found to be larger innodules on two Morella species than in nodules formed onthree Elaeagnus and one Shepherdia species after inocula-tion with the same soil slurry [11]. In addition, thedistribution of Frankia populations and their abundance innodules was unique for each of the plant species and noneof them captured the entire diversity of frankiae retrievedfrom this soil. All of these studies were based on bioassaysin which specific capture plants were inoculated with soilslurries and formed nodules analyzed for abundance ordiversity of frankiae [9–11]. The choice of the capture plantspecies in bioassays thus has significant effects onabundance and diversity estimates of frankiae in soil.Consequently, bioassays with a single-plant species ascapture plant do not provide an accurate quantitative pictureof the overall structure of nodule-forming populations insoil.

Comparative analyses of gene clone libraries generatedfor several soils with sequences obtained from nodulesformed in bioassays with the same soils displayed a muchlower diversity in clone libraries, and large differencesbetween sequences retrieved from clone libraries and thoseobtained from nodules, with assignments to the samephylogenetic group only rarely encountered for individualsoils [12]. Assuming that the low diversity of sequences ingene clone libraries was due to preferential amplification ofmore abundant sequences in these soils [13], gene clonelibrary analyses should reflect an accurate assessment of themost abundant Frankia populations. These results againindicate the inadequacy of bioassays for quantitativeassessments of total Frankia populations in soils. Theyalso raise the question whether bioassay analyses providean accurate picture of nodule-forming frankiae in soil, orwhether they reflect artifacts due to the experimentalmanipulations.

The aim of this study was therefore to eliminate theexperimental manipulation step and analyze soil and rootnodule populations of frankiae in different actinorhizalplant species growing sympatrically in nature. The studyincluded strain-level diversity analyses of frankiae in rootnodules of different alder species and subspecies growingin the same soil to assess intrageneric effects of plantspecies with respect to nodule-formation by Frankia strainsfrom soil. This assessment was followed by comparativesequence analyses of nifH gene fragments of frankiae

retrieved from selected root nodules to a correspondinggene clone library from the soil in order to evaluatepotential selection of frankiae by plants compared toabundance in soil. Additional studies included a compara-tive analysis of Frankia populations in root nodules ofselected Alnus taxa growing on different soils in order toinvestigate potential differences in nodule-formingpopulations of frankiae.

Materials and Methods

Nodule and Soil Sampling

Root nodules were collected from twelve alder taxa, i.e. A.incana subsp. incana, Alnus japonica, A. glutinosa, A.incana subsp. tenuifolia, A. incana subsp. rugosa, Alnusrhombifolia, Alnus mandshurica, Alnus maritima, Alnusserrulata, A. incana subsp. hirsuta, A. glutinosa var.pyramidalis, and A. incana subsp. rugosa var. americanagrowing sympatrically in the Morton Arboretum in Lisle,IL, USA (41° 48′ 52.5′′ N, 88° 04′ 15" W). The soil (#107)belongs to the Sawmill series that consists of very deep,generally poorly drained soils formed in alluvium on floodplains (http://www.2.ftw.nrcs.usda.gov/osd/dat/S/SAW-MILL.html). Additional nodules were obtained fromselected alder species growing on soils other than soil#107 in the arboretum. A. glutinosa also occurred on soil#1107, a wetter version of soil #107, and in soil #534, aman-made clayey soil from earthmoving for road construc-tion, while A. incana subsp. hirsuta was growing in soil#194 of the Ozkaukee soil series (http://www.2.ftw.nrcs.usda.gov/osd/dat/html) and A. incana subsp. rugosa var.americana in soils #1107, #194, and in soil #531 of theMarkham soil series (http://www.2.ftw.nrcs.usda.gov/osd/dat/html; Table 1). A surface sample of soil #107 (down toa depth of about 10 cm) was collected under an individualof A. glutinosa, sieved (mesh size 4.5 mm) and stored at 4°C until further use.

Characterization of Frankiae in Root Nodules

DNA extraction Ten nodules were selected from oneindividual tree of each of the alder taxa growing on soil#107. For seven of these taxa, additional individuals wereavailable (Table 1) and nodules from these trees wereincluded in the analyses, as were additional nodulesobtained from selected alder taxa growing on other soilsthan soil #107 in the arboretum. For DNA extraction, asingle lobe of each nodule was surface sterilized in 30%H2O2 for 10 min, subsequently washed three times withsterile water, and the epidermis removed. Remaining tissuewas homogenized with a mortar and pestle in 1 ml of sterile

Frankia Populations in Soil and Root Nodules of Sympatrically Grown Alnus Taxa 93

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water, and the homogenates were transferred to anEppendorf tube and centrifuged at 14,000×g for 1 min.Pellets were washed once with 0.1% sodium pyrophosphatein water (wt/vol), followed by two washes with steriledistilled water. Subsequently, the nodule pellets as well asthe pellets of pure cultures (approximately 50 mg) used forcomparison, were re-suspended in 95 μl of distilled water,mixed with 5 μl of proteinase K solution (Promega,Madison, WI, USA; 30 U mg−1, 10 mg ml−1 in water)and incubated at 37°C for 20 min. After that, 0.5 μl of a

10% SDS solution was added and the mixture wasincubated at 37°C for another 3 h which was followed bya final incubation at 80°C for 20 min. Nucleic acids weresubsequently purified from the lysates using the GenScriptQuickClean DNA Gel Extraction Kit (GenScript, Piscat-away, NJ,USA), and resuspended into a final volume of30 μl.

Rep-PCR Rep-polymerase chain reaction (PCR), a PCR-assisted fingerprinting technique targeting consensus motifs

Table 1 Alnus taxa sampled for the analyses of frankiae in root nodules by rep-PCR and nifH gene sequence analyses

Soil Series (basic description)

Alnus taxa Age (years)a Rep-PCR profileb Sequence acronym

107 Sawmill Series Cumulic Endoaquoll (silty clay loam, mixed, superactive, mesic, very deep, poorly drained, pH 7.4–7.8)

A. incana subsp. incana 26 I

25 I (n=5)

A. japonica 47 I

A. glutinosa 26 I

26 I (n=9)

10 I (n=7)

A. incana subsp. tenuifolia 30 I Atnod107

30 I (n=4)

A. incana subsp. rugosa 22 I

22 I (n=5)

83 (plant) I (n=5)

A. rhombifolia 17 I

A. mandshurica 5 I

A. maritima 9 I

A. serrulata 26 I

A. incana subsp. hirsuta 21 II

21 (plant) II (n=10)

A. glutinosa var. pyramidalis 48 (graft) II Agpnod107

18 (cutting) II (n=4)

A. incana subsp. rugosa var. americana 24 III Arvnod107

24 III (n=5)

194 Ozkaukee Series Oxyaquic Hapludalf (silt loam, illitic, mesic, moderately deep, moderately well drained, pH 6.6–7.3)

A. incana subsp. hirsuta 27 nd Ahnod194

A. incana subsp. rugosa var. americana 24 nd Arvnod194

531 Markham Series Mollic Oxyaquic Hapludalf (silt loam, illitic, mesic, very deep, moderately well drained, pH 6.6–7.3)

A. incana subsp. rugosa var. americana 24 nd Arvnod531

534 Excavated glacial till and loess Typic Udorthent (fine-grained minerals from earthmoving for road construction, well drained, pH 6.6–7.3)

A. glutinosa 11 nd Agnod534

1107 Sawmill Series Cumulic Endoaquoll (silty clay loam, mixed, superactive, mesic, very deep, very poorly drained, pH 7.4–7.8)

A. glutinosa NA nd Agnod1107

A. incana subsp. rugosa var. americana 24 nd Arvnod1107

NA not available, nd not determineda Plants were grown from seeds unless mentioned otherwiseb n=10 nodules per individual tree unless mentioned otherwise

94 A. Pokharel et al.

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of repetitive elements common to prokaryotic genomeswas performed in a total volume of 25 μl containing4 μl of 5× Gitschier buffer (83 mM (NH4)2SO4,33.5 mM MgCl2, 335 mM Tris/HCl, pH 8.8, 33.5 μMEDTA, 150 mM β-mercaptoethanol), 1.25 μl dNTPs(100 mM each, mixed 1:1:1:1), 2.5 μl di-methyl-sulfoxide (DMSO), 0.2 μl bovine serum albumin (BSA,20 mg ml−1), 1.3 μl of primer BoxA1R (5′CTA CGG CAAGGC GAC GCT GAC G; 300 ng μl−1) [14], 2 μl ofpurified DNA and 0.4 μl Taq polymerase (5 U μl−1) [15].Taq polymerase was added after an initial incubation at96°C for 10 min. After an additional denaturation at 95°Cfor 2 min, 30 rounds of temperature cycling in a PTC-200Thermocycler (MJ Research, Waltham, MA, USA) withdenaturation at 94°C for 3 s and subsequent 92°C for 30 s,primer annealing at 50°C for 1 min, and elongation at 65°C for 8 min were performed. This was followed by a finalelongation at 65°C for 8 min [15, 16]. Profiles wereanalyzed by gel electrophoresis on 2% agarose gels inTAE buffer after staining with ethidium bromide(0.5 μg ml−1) [17].

NifH gene sequence analysis NifH gene fragments(606 bp) from frankiae in selected root nodules wereamplified using primers nifHf1 (5′GGC AAG TCC ACCACC CAG C3′) and nifHr (5′CTC GAT GAC CGT CATCCG GC3′) in a reaction volume of 50 μl, containing 1 μlof a 10 mM dNTP mix, 0.5 μl each primer (0.4 μM),8.2 μl BSA (30 μg ml−1), 5 μl of 10× PCR buffer with15 mM MgCl2, 2 μl DNA from root nodules or from purecultures, and 0.2 μl Taq DNA polymerase (5 U μl−1; GeneScript, Piscataway, NJ, USA). Taq polymerase was addedafter an initial incubation at 96°C for 10 min. The additionof Taq polymerase was followed by 35 rounds oftemperature cycling (96°C for 30 s, 60°C for 30 s, and72°C for 45 s) and final 7-min incubation at 72°C. Sub-samples of the reactions (10 μl) were checked foramplification products by gel electrophoresis (2% agarosein TAE buffer, wt/vol) after staining with ethidiumbromide (0.5 μg ml−1) [17].

Amplicons with appropriate sizes were purified usingthe Ultra Clean 15 DNA Purification Kit (MoBio,Carlsbad, CA, USA), and sequenced using the CEQ8800 Quickstart Kit according to the manufacturer’sinstructions (Beckman Coulter, Fullerton, CA, USA)with the addition of 5% DMSO to the reaction mix. Thesequencing reaction consisted of an initial incubation at76°C for 5 min followed by 76°C for 5 min duringwhich primer and master mix were added, a subsequentincubation at 94°C for 2 min, and 35 cycles oftemperature cycling (94°C for 30 s, 50°C for 30 s,and 60°C for 4 min) and a final extension at 60°C for10 min [18]. Sequences were analyzed on a CEQ 8800

capillary action sequencer (Beckman Coulter), andsubmitted to GenBank under accession numbersGQ884048 to GQ884062, GQ884122, GQ884141, andGQ884142.

Characterization of Frankiae in Soil

DNA extraction DNA was extracted from two 0.5-gsamples of soil #107 after cells were disrupted by agitationin a Mini-Bead-Beater-8 (BioSpec Products, Inc, Bartles-ville, OK, USA) for 2 min. Beads, soil, and cell debris wereseparated from extraction buffer by centrifugation at14,000 rpm for 1 min. Nucleic acids released into thebuffer were purified by sequential phenol, phenol/chloro-form, and chloroform extraction [17], followed by precip-itation with two volumes of 2.5 M NaCl/20% PEG 8000[19]. After an additional extraction with phenol/chloroformand subsequent mixing of the supernatant with one volumeof isopropanol, nucleic acids were precipitated by centrifu-gation at 14,000 rpm for 10 min, washed with 70% ethanol,air dried, and finally re-suspended in 30 μl of steriledistilled water.

Nucleic acids of both extractions were pooled and2 μl of this solution were used as template to amplifynifH gene fragments (606 bp) using primers nifHf1 andnifHr in a reaction volume of 50 μl as described above.The product of this PCR reaction that was generallyinvisible when analyzed by gel electrophoresis waspurified using the Ultra Clean 15 DNA Purification Kit(MoBio, Carlsbad, CA, USA) into 20 μl, and diluted to200 μl with distilled sterilized water. One microliter of thediluted PCR product was used as template in a nested PCRreaction, using the same conditions as above, except thatprimer nifHr269 (5′CCG GCC TCC TCC AGG TA) wasused as reverse primer instead of nifHr. PCR reactionswere analyzed for amplicons of a size of about 270 bp bygel electrophoresis (2% agarose in TAE), which were thenisolated from the gel using the Ultra Clean 15 DNAPurification Kit (MoBio). Purified fragments were ligatedinto pGEM®-Teasy (Promega) according to the manufac-turer’s instructions, and transformed into Escherichia coliTOP-10 (Stratagene, Cedar Creek, TX, USA). Cloneswere analyzed at random for partial nifH gene fragmentsby PCR using the conditions described above for rootnodule analyses except that primers seqf (5′TCA CACAGG AAA CAG CTA TGA C) and seqr (5′CGC CAGGCT TTT CCC AGT CAC GAC) were used foramplification. These primers were also used for sequenceanalyses of the resulting amplicons, with the remainingconditions being identical to those described above foramplicons from root nodules. Sequences were submittedto GenBank with accession numbers GQ884063 toGQ884121, and GQ884123 to GQ884140.

Frankia Populations in Soil and Root Nodules of Sympatrically Grown Alnus Taxa 95

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Phylogenetic Analyses

Sequences of nifH gene fragments obtained from frankiaefrom root nodules of plants (n=18), those from purecultures of Frankia (n=23), and those obtained from thesoil gene clone library (n=73) were trimmed to be 269 bplong and aligned using Sequencher 4.2.2 (Gene CodesCorp., Ann Arbor, MI, USA), CLUSTAL X and MacClade4.05 [20, 21] and analyzed using maximum parsimony,neighbor joining, Bayesian, and maximum likelihood (ML)methods as described in detail in previous studies [11, 22].For presentation purposes, this dataset was split into twodatasets: The first dataset contained sequences representinguncultured frankiae in the root nodules of twelve aldergenotypes growing in soil #107, sequences of frankiae fromthe soil clone library and those of pure cultures, while thesecond dataset contained sequences of uncultured frankiaein root nodules of all alder types growing in soils #107,#1107, #194, #531, and #534 and those of pure cultures.

Results

Rep-PCR analyses of uncultured Frankia populations inroot nodules of 12 Alnus taxa (n=10 nodules each) growingsympatrically in the Morton Arboretum revealed identicalpatterns for nodules from each Alnus taxon, includingreplicate trees of the same taxon, and low diversity overallwith only three profiles retrieved (Fig. 1; Table 1). Oneprofile was retrieved from all nodules of nine host types(profile I: A. incana subsp. incana, A. japonica, A.glutinosa, A. incana subsp. tenuifolia, A. incana subsp.rugosa, A. rhombifolia, A. mandshurica, A. maritima, andA. serrulata), the second was found in all nodules of twoplant taxa (profile II: A. incana subsp. hirsuta and A.glutinosa var. pyramidalis), and the third was unique for allFrankia populations in nodules of A. incana subsp. rugosavar. americana (profile III). These three rep-PCR profilesdiffered from those of Frankia strains Ag45/Mut15 andArI3 used as positive controls, with no amplificationdetected in the negative controls.

Comparative sequence analyses of nifH gene fragmentsfrom frankiae in nodules of three Alnus types representingeach rep-profile (i.e., A. incana subsp. tenuifolia (profile I),A. glutinosa var. pyramidalis (profile II), and A. incanasubsp. rugosa var. americana (profile III), respectively,present with several individuals in soil #107) revealedsequence similarities between nifH gene fragments fromfrankiae in nodules of A. incana subsp. rugosa var.americana (Arvnod107) to that of A. incana subsp.tenuifolia (Atnod107) of about 94%, and to frankiae innodules of A. glutinosa var. pyramidalis (Agpnod107) ofabout 97%. Independent of the phylogenetic method

employed for analyses, identical assignments of theseFrankia populations in nodules to pure cultures of frankiaewere obtained. For presentation purposes only, the ML treewas used to demonstrate the relationship between purecultures and root nodule Frankia for each plant analyzed(Fig. 2). Frankiae in nodules of A. incana subsp. tenuifoliaand A. incana subsp. rugosa var. americana clustered withFrankia strains of subgroup AII (represented by strainAg45/Mut15) and subgroup AI (represented by strainArI3), respectively, with high bootstrap support measures(BS) and posterior probabilities (PP) values indicating thestability of these groups (Fig. 2). The position of strainAgpnod107 from A. glutinosa var. pyramidalis, however,was inconsistent and not supported by BS and PP measures(Figs. 2, 3). This inconsistency was most likely due to long-branch attraction, which affects the reliable assignment ofsequences in the absence of closely related ingroupsequences as discussed previously [12]. The inclusion ofsequences from uncultured frankiae did not resolve thisproblem because the sequence of Agpnod107 showed only97% sequence similarity to that of sequences of its closestrelatives, strain ArI3 and an uncultured Frankia from soil(clone AO3-14nodF, Accession #FJ977313).

Sequence analyses of 73 randomly selected clones fromthe nifH gene clone library of soil #107 identified allsequences retrieved as representing frankiae of the Alnus

A. glutin

osa

A. inca

na subsp

. rugosa

A. inca

na subsp

. tenuifo

lia

A. mandsch

urica

A. marit

ima

A. inca

na subsp

. inca

na

A. japonica

A. rhombifo

lia

A. serr

ulata

A. inca

na subsp

. hirs

uta

A. glutin

osavar.

pyramyd

alis

A. inca

na subsp

. rugosa

564

2027

2322

var. ameri

cana

* **

I II IIIRep-PCR profile

Figure 1 Rep-PCR profiles of Frankia in the root nodules ofdifferent Alnus taxa. The first nine patterns are identical to each other(profile I), third and second from the last are identical (profile II) andthe last pattern is unique (profile III). Names on the top are of the aldertaxon from which the corresponding profiles were generated.Fragment sizes on the left represent those of a Lambda HindIIIDNA size marker

96 A. Pokharel et al.

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host infection group, with all sequences being almostidentical and clustering together in subgroup AI (Fig. 2).None of these sequences was identical to any of thoseobtained from frankiae in nodules; they were, however,very similar to those representing frankiae in nodules of A.incana subsp. rugosa var. americana (Arvnod107) withsequence similarities of up to 99.7% (Fig. 2).

Since A. incana subsp. tenuifolia and A. glutinosa var.pyramidalis were only present on soil #107, A. glutinosaand A. incana subsp. hirsuta were used instead in additionalsequence analyses of nifH gene fragments of Frankiapopulations in nodules formed on different soils. On soil#107, the taxa harbored the same Frankia strains in nodulesas A. incana subsp. tenuifolia and A. glutinosa var.pyramidalis, respectively. Together with A. incana subsp.rugosa var. americana, these taxa represented the threestrains characterized by rep-PCR profiles I, II, and III.Comparative sequence analyses of nifH gene fragmentsrevealed the presence of different Frankia populations innodules for each soil, with populations showing identicalsequences in nodules from the same soil, but differencesbetween plant species. Frankiae in root nodules of A.glutinosa growing in soils #107, #534 and #1107 (Atnod107,

Agnod534 and Agnod1107, respectively), for example, wererepresented by three sequences, with sequences Atnod107and Agnod1107 clustering in subgroup AII and those fromAgnod534 in subgroup AI within the Alnus host infectiongroup (Fig. 3). NifH sequences of frankiae in nodules of A.incana subsp. hirsuta in soils #107 and #194 (Agpnod107and Ahnod194) differed from each other, however, clusteredin the same subgroup AI (Fig. 3). Similar results wereobtained for nifH gene sequences Arvnod107, Arvnod194,Arvnod531 and Arvnod1107 of frankiae in nodules of A.incana subsp. rugosa var. americana growing in soils #107,#194, #531 and #1107 (Fig. 3).

Discussion

Rep-PCR that was used as a reliable tool to distinguishindividual strains in root nodules of different Alnusgenotypes demonstrated the presence of only one Frankiastrain in nodules of each taxon including replicate treesgrowing on the same soil. Although these results mighthave been impacted by the limited number of nodulesanalyzed (i.e., ten nodules per plant taxon, plus an

AgP1R3 (FJ477428)AgP1R2 (FJ477427)AgP1R1 (FJ477426)

AvcI1 (EU862916)ACN14a (EU862907)

AvsI4 (FJ477436)ArI3 (EU862915)

CpI1 (FJ477438)AgKG’84/4 (EU862909)AgKG’84/5 (FJ477424)

AgB20 (FJ477440)AgB32 (FJ477421)AgB16 (FJ477439)

AiPs3 (FJ477441)AgGS’84/18 (FJ477423)

Ag45/Mut15 (EU862908)CeF (FJ477437)

CjI-82 (EU862919)CcI3 (EU862918)

HrI1 (EU862922)EAN1pec (EU862921)

0.01

-(93,100,89)

100 (99,99,88)

100(100,100,100)95(90,100,88)

100(100,100,100) 100(100,100,98)

Arvnod107 (GQ884122)

Atnod107 (GQ884054)

AI

AII

AIIIEI

AgGS’84/44 (FJ477422)

100(100,100,100)

Agpnod107 (GQ884142)

Clones from soil (GQ884063-884121 andGQ884123-884140)

Ea1.12 (EU862920)

III

II I

Figure 2 Maximum likelihoodtree based on comparative se-quence analysis of nifH genefragments showing the phyloge-netic position of unculturedFrankia populations in rootnodules of Alnus taxa fromsoil #107 within subgroups ofthe Alnus host infection group(AI–III). Numbers reflectbootstrap support measures(BS), and numbers in paren-theses represent BS measuresand posterior probabilities (PP)from maximum parsimony,Bayesian, and neighbor-joining analyses, respectively.Sequences representing strainsof the Elaeagnus host infectiongroup (i.e., subgroup EI) wereused as out group. The grayoval areas highlight the posi-tion of the sequences of frank-iae from root nodules (nod) ofthe respective Alnus type insoil #107 (At A. incana subsp.tenuifolia; Agp A. glutinosavar. pyramidalis, Arv A. incanasubsp. rugosa var. americana),and adjacent numbers identifythe corresponding rep-PCRprofiles I, II, or III

Frankia Populations in Soil and Root Nodules of Sympatrically Grown Alnus Taxa 97

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additional two to three for replicate trees of a few selectedAlnus types) the fact that the same phenomenon was foundfor all 12 Alnus taxa, and with three different Frankiastrains, suggests a highly selective interrelationship be-tween nodule-forming frankiae in soil and the host planttaxon in an established alder stand. These results are incontrast to those of other studies that found differentFrankia strains in nodules of the same host plant undernatural conditions, even though overall diversity wasfound to be low [23, 24]. Bioassay analyses generallyresult in the detection of a much higher diversity innodules formed on individual capture plant species [11,22] indicating that they are affected by experimentalmanipulations, and thus do not retrieve information onnodule-forming frankiae established long-term in a partic-ular soil.

Three Frankia strains representing distinct groups withinthe Alnus host infection group were found to form nodules,with individual strains forming all nodules analyzed on aparticular Alnus taxa. These results indicated a large effectof the host plant taxon in the selection of Frankia strains forroot nodule formation, assuming that soil environmentalconditions were identical within an Alnus stand. Specificconditions at each nodule collection site, even though thesoil type was the same, were not determined, and thus theeffects of variable soil environmental conditions within asoil-survey type on nodule-forming frankiae cannot beentirely excluded for any of the Alnus genotypes. Ourresults, however, corroborate conclusions of others that hostplant species are a significant variable that can distinguishFrankia populations for nodule formation at a particularsite [25].

The three populations representing frankiae in allnodules formed on the 12 Alnus species, subspecies orvarieties growing on soil #107, were assigned to differentsubgroups within the Alnus host infection groups. Asoutlined in previous studies [11, 22], these subgroupsresemble clusters with 95−97% similarity values of thenifH gene sequences, which correlate largely to genomicgroupings established previously in DNA−DNA relatednessstudies [26–31]. All three strains are represented by uniquenifH gene sequences distinct from those of all isolatesanalyzed so far. While the position of strain Agpnod107 thatrepresents 17% of the frankiae in nodules can be consideredas being ambiguous, strain Atnod107, that represents 75% ofthe frankiae in nodules is clearly clustering within subgroupAII. Subgroup AII harbors Frankia isolates characterizedby their ability to grow on complex organic matter such asleaf litter whereas those belonging to subgroup AI to whichstrain Arvnod107 (8% of frankiae in nodules) was assigneddid not grow on complex organic matter [32, 33]. It istempting to use these phylogenetic assignments to speculateabout potential physiological differences in the develop-ment of Frankia populations in soils, e.g., with thoserepresenting subgroup AII growing better in soils, conse-quently being more abundant and thus forming morenodules. However, in addition to effects of plant selectivity,several uncertainties prevent any serious discussion of thisissue. The uncertainties include questions on the occurrenceof the infectious Frankia particles in soil, which could beactively growing organisms present in a vegetative form (i.e., in long filaments as in pure culture, in short fragments orin single-cell form) or inactive forms such as spores that areactivated by exudates of the host plant [12].

It has been speculated that the presence of a Frankiastrain in nodules is positively related to its abundance insoil [23]. Our results contradict this speculation becausenifH gene clone library analysis retrieved only sequencesrepresenting frankiae distantly related to those in nodules,

Arvnod351a (GQ884060)Arvnod351b (GQ884059)

Arvnod107 AgKG’84/4AgKG’84/5

AgB20AgB32AgB16

Arvnod1107a (GQ884057)Arvnod1107b (GQ884058)

Ahnod194a (GQ884055)ACN14a

AgP1R3Arvnod194a (GQ884056)Arvnod194b (GQ884141)AgP1R2AgP1R1

AvcI1AvsI4ArI3

CpI1Agpnod107

AiPs3 AgGS’84/18Ag45/Mut15AgGS’84/44

CeFCcI3CjI-82

HrI1EAN1pec

EuI1c

99(100,98,100)

100(100,100,100)

100 (100, 100, 100)

100(100,100,100)

100(100,100,100)

99(100,98,100)

97(100,-,-)

0.01

70(-,-,100)

-(94,100,100)

97(94,92,100)

-(99,-,100)

-(94,85,100)

-(-,70,100)

97(100,83,98)

-(78,81,99)

AI

AII

AIII

EI

Agnod534a (GQ884062)Agnod534b (GQ884061)

Agnod1107e (GQ884052)Agnod1107f (GQ884051)

Agnod1107de (GQ884049)Agnod1107b (GQ884048)

Agnod1107c (GQ884053)

Agnod1107a (GQ884050)

Atnod107

Figure 3 Maximum likelihood tree based on comparative sequenceanalysis of nifH gene fragments showing the phylogenetic position ofuncultured Frankia populations in root nodules of A. glutinosa (Ag,instead of A. incana subsp. tenuifolia (At)), A. incana subsp. hirsuta(Ah, instead of A. glutinosa var. pyramidalis (Agp)) and A. incanasubsp. rugosa var. americana (Arv), representing the three rep-profileswhen growing on soil #107 from different soils (#107, #1107, #194,#351, and #534) within subgroups of the Alnus host infection group(AI–III). Numbers reflect bootstrap support measures (BS), andnumbers in parentheses represent BS measures and posterior proba-bilities (PP) from maximum parsimony, Bayesian and neighborjoining analyses, respectively. Sequences representing strains of theElaeagnus host infection group (i.e., subgroup EI) were used as outgroup

98 A. Pokharel et al.

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with sequences that were least abundant in nodules (i.e.,Arvnod107 representing 8% of frankiae in nodules) beingthe most similar to those from soil. This result iscomparable to that of previous analyses in which nodule-forming frankiae retrieved in bioassays were not accuratelyrepresented by sequences obtained in gene clone librariesfrom soils [12]. It is noteworthy that the host plant typewith nodular Frankia strains most similar to those found inassociated soils was A. incana subsp. rugosa var. america-na, a taxon in the study that is native to the region.Statements that root nodule populations do not represent themost abundant Frankia strains in soil or that host plantsselect a particular Frankia strain for root nodule formationregardless of its abundance in soil, however, require carefulconsideration because they rely on gene clone librariesproviding an accurate picture of the diversity and abun-dance of frankiae in soil. Since our analysis is based on onelibrary only, a larger set of libraries from replicate samplesand a higher number of sequences are necessary to accountfor variation between samples in order to obtain a morecomprehensive picture. Although it has been shown inseveral studies on other microorganisms that gene clonelibraries can represent the abundance of target organismsadequately [13, 34, 35], the opposite has been shown aswell [36–38]. Thus, any statements on the abundance ofspecific Frankia populations in soil based upon our libraryanalysis remain highly speculative.

Soil characteristics can affect nodule formation by Frankiapopulations [39–44]. Considering the differences in pH, soilmoisture regimes, the matric potential, or other physico-chemical properties of the soils, it is not surprising thatdifferent Frankia populations were found in nodules of Alnusspecies and subspecies growing on different soils, with strainsbeing different in nodules of different Alnus genotypesgrowing on the same soil, or in nodules of the same Alnusgenotype growing on different soils. These results confirm theinfluence of plant-host genetics on the selection of a particularFrankia strain for root nodule formation, but also indicateeffects of environmental conditions on nodule-formingfrankiae in each soil. While these results provide novelinformation on the selectivity of multiple plant genotypeswithin a genus for soil frankiae nodulation under specificedaphic conditions, additional studies will be required toresolve the issues concerning the abundance and occurrenceof infectious Frankia particles in soil (i.e., active vs. inactive,filamentous vs. single celled). Continued elucidation of thecomplex symbiotic interactions of actinorhizal partners in soilcould lead to the maintenance and improvement of nitrogen-based productivity in managed ecosystems.

Acknowledgments The authors thank Pamela Frederick for obtain-ing nodule samples and the Morton Arboretum for permission tocollect plant and soil materials for this study.

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