Diversity of frankiae in root nodules of Morella pensylvanica grown in soils from five continents

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Abstract

Bioassays with Morella pensylvanica as capture plant and comparative sequence analyses of nifH gene fragments of Frankia populations in nodules formed were used to investigate the diversity of Frankia in soils over a broad geographic range, i.e., from sites in five continents (Africa, Europe, Asia, North America, and South America). Phylogenetic analyses of 522-bp nifH gene fragments of 100 uncultured frankiae from root nodules of M. pensylvanica and of 58 Frankia strains resulted in a clear differentiation between frankiae of the Elaeagnus and the Alnus host infection groups, with sequences from each group found in all soils and the assignment of all sequences to four and five clusters within these groups, respectively. All clusters were formed or dominated by frankiae obtained from one or two soils with single sequences occasionally present from frankiae of other soils. Variation within a cluster was generally low for sequences representing frankiae in nodules induced by the same soil, but large between sequences of frankiae originating from different soils. Three clusters, one within the Elaeagnus and two within the Alnus host infection groups, were represented entirely by uncultured frankiae with no sequences from cultured relatives available. These results demonstrate large differences in nodule-forming frankiae in five soils from a broad geographic range, but low diversity of nodule-forming Frankia populations within any of these soils.

Introduction

Actinorhizal plants are characterized by their ability to form root nodules in symbiosis with the nitrogen-fixing actinomycete Frankia, which enables them to grow on sites with restricted nitrogen availability [2]. They therefore resemble typical pioneer plants that frequently establish themselves after flooding, fires, landslides, glacial activity, as well as volcanic eruptions [13]. Actinorhizal plants are nearly ubiquitously distributed world-wide, and their occurrence in diverse habitats like deserts and swamps, forests and beaches, at high or low elevations and in many other places between these extremes has been well described (see [6] for review). Information on the geographic distribution and diversity of frankiae, the symbiotic partner of actinorhizal plants, is much more limited, and usually restricted to frankiae in root nodules on specific host plants at a particular site rather than on populations in soil analyzed over a large geographic range [5], [31], [32].

Root nodules represent a natural locale of enrichment of one Frankia strain that can easily be characterized by a large variety of methods such as comparative sequence analyses of 16S rRNA genes [10], [46], [56], or genes like glnII [11], [22] or nifH [21], [38], but also by highly distinctive tools such as Rep-PCR [37], [47], [48], or PCR-RFLPs [36], [40], [50] that generally require the availability of pure cultures. The usefulness of these tools is largely reduced in soils that represent highly heterogeneous environments with a tremendous diversity of organisms [16], [20], [67], [68] and with frankiae present in low numbers (approximately 104–105 cells g−1 soil) [23], [49] as part of a large microbial community (more than 109 cells g−1 soil) [8], [71]. PCR-based methods, distinctive target genes (e.g., 16S rRNA gene), and specific primers are available for the detection of frankiae on the genus level [24]; however, more specific analyses within the genus are hampered by the low abundance of frankiae in soil and the limited resolution of phylogenetically relevant target genes such as rRNAs for assessing diversity within the genus.

In a recent study we used comparative sequence analyses of nifH gene fragments to distinguish Frankia populations in different root nodules on the same plant species, and to analyze the diversity of Frankia populations from the same soil forming nodules on different host plant species [45]. The study used plant bioassays, i.e., actinorhizal plants inoculated with dilutions of soil slurries, in order to reduce potential effects of physical and chemical characteristics of the soil on nodule formation. This study demonstrated large host plant effects on the selection of frankiae for root nodule formation as well as a large diversity of nodule-forming Frankia populations in one soil that could be distinguished by comparative sequence analyses of nifH gene fragments [45].

In this follow-up study, we used the same basic experimental setup including bioassays and comparative sequence analyses of nifH gene fragments of Frankia populations in nodules formed, to investigate the diversity of Frankia in soils over a broad geographic range spanning five continents. The bioassay used Morella pensylvanica as the capture plant because it is known to be highly promiscuous, harboring a wide diversity of Frankia in root nodules [4], [14], [28]. Additionally, in our previous study M. pensylvanica formed nodules with more diverse Frankia populations of the Elaeagnus host infection group than different Elaeagnus plant species, and also with Frankia of the second major host infection group, the Alnus host infection group [45]. In order to adequately assign uncultured frankiae in root nodules to host infection groups and subgroups, additional analyses focused on retrieving nifH gene fragment sequences from a large variety of Frankia strains previously isolated from nodules of plants belonging to either the Alnus or the Elaeagnus host infection group.

Section snippets

Soil collection

Soils were collected in October 2006 from four plots (10 m×10 m, spaced every 300 m along a randomly located 900 m transect) at sites located in 5 continents (Africa [Rwanda], Europe [Hungary], Asia [Japan], North America [Alaska], and South America [Peru]) (Table 1). From each of the four plots, twenty 10-cm deep soil subsamples were collected, pooled, and homogenized, for a total of four pooled samples (referred to as A, B, C, and D). Between each subplot, soil sampling equipment was sterilized

Results and discussion

PCR products of nifH gene fragments were obtained from nodules of M. pensylvanica with all soils and subplots tested, however, only in about 50% of the nodules analyzed. The failure to retrieve products from all nodules might be due to a variety of issues. Nodules were extremely small (1–2 mm in diameter) which impacted the accurate separation of periderm tissue with potentially contaminating organisms from nodule tissue with frankiae, and thus frankiae could have been removed unintentionally in

Acknowledgements

The authors thank Dr. Laura G. Perry and the Restoration Ecology Laboratory Crew (Liza Bodistow, Bryan Brown, Jeremy Buss, Chris Herron, Lilly Hines, Tim Hoelzle, Ben Hoffman, Erin Klamper, Katie Legg, Mandy Roesch, Travis Talbot, Hannah Varani, and William Vieth) for propagation, maintenance, and harvesting of greenhouse plants. The authors are indebted to the Texas State Department of Biology and the National Science Foundation (GK-12 Grant no. 0742306).

References (71)

  • R. Rossello-Mora et al.

    The species concept for prokaryotes

    FEMS Microbiol. Rev.

    (2001)
  • D.J. Wolters et al.

    Ineffective Frankia strains in wet stands of Alnus glutinosa L. Gaertn. in the Netherlands

    Soil Biol. Biochem.

    (1997)
  • A.D.L. Akkermans et al.

    The family Frankiaceae

  • C.S. An et al.

    Relationships of Frankia isolates based on deoxyribonucleic acid homology studies

    Int. J. Syst. Bacteriol.

    (1985)
  • D.D. Baker

    Relationships among pure cultured strains of Frankia based on host specificity

    Physiol. Plant

    (1987)
  • J.M. Batzli et al.

    Distribution and abundance of infective, soilborne Frankia and host symbionts Shepherdia, Alnus, and Myrica in a sand dune ecosystem

    Can. J. Bot.

    (2004)
  • D.R. Benson et al.

    Recent advances in the biogeography and genecology of symbiotic Frankia and its host plants

    Physiol. Plant

    (2007)
  • A. Capellano et al.

    Root-nodules formation by Penicillium sp. on Alnus glutinosa and Alnus incana

    Plant Soil

    (1987)
  • M. Chavez et al.

    Genetic diversity of Frankia microsymbionts in root nodules from Colletia hystrix (Clos.) plants by sampling at a small-scale

    World J. Microbiol. Biotechnol.

    (2006)
  • M.L. Clawson et al.

    Typical Frankia infect actinorhizal plants exotic to New Zealand

    New Zeal. J. Bot.

    (1997)
  • M.L. Clawson et al.

    Dominance of Frankia strains in stands of Alnus incana subsp. rugosa and Myrica pensylvanica

    Can. J. Bot.

    (1999)
  • S.V. Dobritsa et al.

    Infectivity and host specificity of strains of Frankia

    Microbiology (New York)

    (1990)
  • S.V. Dobritsa et al.

    Genome identity of different Nocardia autotrophica isolates from Alnus spp. root nodules and rhizosphere

  • J. Dunbar et al.

    Empirical and theoretical bacterial diversity in four Arizona soils

    Appl. Environ. Microbiol.

    (2002)
  • J. Felsenstein

    Confidence limits of phylogenies: an approach using the bootstrap

    Evolution

    (1985)
  • M.P. Fernandez et al.

    Deoxyribonucleic acid relatedness among members of the genus Frankia

    Int. J. Syst. Bacteriol.

    (1989)
  • G.E. Fox et al.

    How close is close: 16S rRNA sequence identity may not be sufficient to guarantee species identity

    Int. J. System. Bacteriol.

    (1992)
  • J. Gans et al.

    Computational improvements reveal great bacterial diversity and high metal toxicity in soil

    Science

    (2005)
  • D. Hahn et al.

    Extraction of ribosomal RNA from soil for detection of Frankia with oligonucleotide probes

    Arch. Microbiol.

    (1990)
  • D. Hahn et al.

    Variable compatibility of cloned Alnus glutinosa ecotypes against ineffective Frankia strains

    Plant Soil

    (1988)
  • X.H. He et al.

    Natural diversity of nodular microsymbionts of Myrica rubra

    Plant Soil

    (2004)
  • D.M. Hillis et al.

    An empirical test of bootstrapping as a method for assessing confidence in phylogenetic analysis

    Syst. Biol.

    (1993)
  • J.B. Huang et al.

    Host range of Frankia endophytes

    Plant Soil

    (1985)
  • J.P. Huelsenbeck et al.

    MRBAYES: Bayesian inference of phylogenetic trees

    Bioinf. Appl. Note

    (2001)
  • J.P. Huelsenbeck et al.

    Bayesian inference of phylogeny and its impact on evolutionary biology

    Science

    (2001)
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