Quantification of Frankia in soil




Samant, Suvidha S.

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The genus Frankia represents nitrogen fixing actinomycetes that form root nodules with more than 200 actinorhizal plant species. Frankia can be found in root nodules of specific host plants, a natural locale of enrichment of usually one Frankia population, and in soils that represent highly heterogeneous environments. Due to their low abundance in soil and difficulties in the isolation of Frankia from soil, molecular tools such as PCR have been evaluated for quantitative assessments of frankiae in soil. PCR-based quantification of Frankia in soils so far employed PCR-MPN using nested or booster PCR, but did not consider more recent technological advancements such as quantitative real-time PCR (qPCR) that has successfully been used for the quantification of other soil microorganisms. The work presented in this Ph.D. dissertation focuses on two basic objectives: (1) to develop detection and quantification methods that allowed us to analyze Frankia populations in soil, and (2) to employ these methods to address questions on the fate of indigenous and introduced frankiae in soils. Work on the first objective resulted in the development of a SYBR Green based qPCR method that quantified clusters 1 and 3 of the actinomycete Frankia in soils by targeting nifH gene sequences (chapter II). Primer nifHr158 was designed to be used as reverse primer in combination with forward primer nifHf1 specifically amplifying a 191-bp fragment of the nifH gene of these Frankia. The primer combination was tested for specificity on selected pure cultures, and by comparative sequence analyses of randomly selected clones of a clone library generated with these primers from soil DNA extracts. After adjustments of DNA extraction conditions, and the determination of extraction efficiencies used for sample normalization, copy numbers of nifH genes representing Frankia of clusters 1 and 3 were quantified in different mineral soils, resulting in cell density estimates for these Frankia of up to 106 cells [g soil {dry weight}] −1 depending on the soil. The study, however, also revealed problems in the application of nifH genes as targets for the quantification of frankiae. Primers developed only detected frankiae of clusters 1 and 3, but not frankiae of cluster 2 or cluster 4, and indications for nifH gene transfer were observed. These issues prompted us to look for other genes that could be used as target in qPCR applications to quantify all members of the genus Frankia but also to distinguish clusters or specific subgroups within the genus. In chapter III, we report on the evaluation of 23S ribosomal RNA gene sequences as potential target for the detection of all members of the genus Frankia and specific subgroups within the genus. A qPCR with a primer combination targeting all nitrogenfixing frankiae (clusters 1, 2 and 3) resulted in numbers similar to those obtained with a previously developed qPCR using nifH gene sequences, both with respect to introduced and indigenous Frankia populations. Primer combinations more specifically targeting three subgroups of the Alnus host infection group (cluster 1) or members of the Elaeagnus host infection group (cluster 3) were specific for introduced strains of the target group, with numbers corresponding to those obtained by quantification of nitrogen-fixing frankiae with both the 23S rRNA and nifH genes as target. Method verification on indigenous Frankia populations in soils, i.e. in depth profiles from four sites at an Alnus glutinosa stand, revealed declining numbers in the depth profiles, with similar abundance of all nitrogen-fixing frankiae independent of 23S rRNA or nifH gene targets, and corresponding numbers of one group of frankiae of the Alnus host infection only, with no detections of frankiae representing the Elaeagnus, Casuarina, or a second subgroup of the Alnus host infection groups. In chapter IV, we report on the application of our qPCR-based quantification method in the assessment of the abundance and diversity of Frankia in four soils with similar physicochemical characteristics, two of which were vegetated with a host plant, Alnus glutinosa, and two with a non-host plant, Betula nigra. Analyses of DAPI-stained cells at three locations, i.e. at a distance of less than 1 m (near stem), 2.5 m (middle crown) and 3-5 m (crown edge) from the stems of both tree species revealed no statistically significant differences in abundance. Frankiae generally accounted for 0.01 to 0.04% of these cells, with values between 4 and 36 x 105 cells (g soil)-1. In three out of four soils, abundance of frankiae was significantly higher at locations “near stem” and/or “middle crown” compared to “crown edge”, while numbers at these locations were not different in the fourth soil. Frankiae of the Alnus host infection group were dominant in all samples accounting for about 75% and more of the cells, with no obvious differences with distance to stem. In three of the soils, all of these cells were represented by strain Ag45/Mut15. In the fourth soil that was vegetated with older A. glutinosa trees, about half of these cells belonged to a different subgroup represented by strain ArI3. In all soils, the remaining cells belonged to the Elaeagnus host infection group represented by strain EAN1pec. Casuarina-infective frankiae were not found. Abundance and diversity of Frankia were similar in soils under the host plant A. glutinosa and the non-host plant B. nigra. Results did thus not reveal any specific effects of plant species on soil Frankia populations shown to have differing nodulation capacities for Alnus in past studies. In chapter V, qPCR was used to follow populations dynamics of indigenous Frankia populations in bulk soil and the rhizosphere of Alnus glutinosa or Casuarina equisetifolia at 2 matric potentials representing “dry” (-0.005 MPa) and “wet” (-0.001 MPa) conditions. Indigenous populations of Frankia in bulk soil (PIATT) that was originally vegetated with Elaeagnus umbellata and had been stored at 4°C for about half a year, increased between 10- and 100-fold within the incubation period of 12 weeks, with usually higher numbers obtained under dry conditions. Abundance of Frankia in the rhizosphere and in bulk soil amended with leaf litter showed a similar pattern, though values for abundance were generally higher, with highest values obtained for Frankia in the rhizosphere of C. equisetifolia. More specific analyses revealed that all frankiae detected at any time and treatment belonged to either subgroup I of the Alnus host infection group or the Elaeagnus host infection group. In bulk soil, growth of frankiae representing the Elaeagnus host infection group was usually faster than that of frankiae of Alnus subgroup I, resulting in higher density increases (i.e. up to 100-fold) than those of Alnus subgroup I (10-fold) during the incubation time. This was different in the rhizosphere of both Alnus and Casuarina plants where effects of matric potential were obtained, with more than 100-fold increases of frankiae of Alnus subgroup I under dry conditions compared to bulk soil at t0, and less than 10-fold increases under wet conditions. The opposite pattern was obtained for frankiae of the Elaeagnus host infection group. Consequently, under dry conditions the genus Frankia in the rhizosphere was to a large extent (i.e. up to 95% depending on the plant species) represented by subgroup I of the Alnus host infection group, while under wet conditions a similar percentage of the genus in the rhizosphere of both plant species was represented by the Elaeagnus host infection group. Leaf litter amendment resulted in growth of frankiae of the Elaeagnus host infection group only, essentially matching the values obtained for genus-specific analyses. Our results demonstrate the usefulness of the qPCR methodology developed in this thesis for ecological studies on frankiae in soils. However, definite conclusions about population dynamics of frankiae in general and individual groups as a function of environmental characteristics require the further reduction of variables (e.g. assessments of population dynamics of individual strains), and the inclusion of additional resources (e.g. soil extracts) in the analyses of population dynamics. Future analyses should also include next generation sequencing techniques using either nifH or rRNA gene fragments as target that should provide insight on overall diversity of frankiae in terrestrial systems and confirm coverage of our qPCR based analyses of all Frankia or specific subgroups.



Frankia, Quantitative PCR, nifH, Root nodules, Clone libraries


Samant, S. S. (2015). <i>Quantification of Frankia in soil</i> (Unpublished dissertation). Texas State University, San Marcos, Texas.


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