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