16S rRNA as molecular marker in ecology of Frankia

The research described in this thesis focusses on the role of biotic factors encountered with the establishment of the symbiosis between black alder plants ( Alnus glutinosa ) and introduced Frankia strains. A selection of plant clones and Frankia strains that gave optimal nodulation and nitrogen fixation in forestry was made. For this reason nodulation tests with increasing complexity were set up. An attempt was made to investigate whether introduced strains behaved differently on plants grown under axenic and non-axenic conditions. Since Frankia strains were difficult to identify by conventional techniques, special attention was given to the development of new molecular techniques for identification of the strains at the nucleic acid level.Inoculation testsInitially, plant material of two physiologically different ecotypes of Alnus glutinosa, the forest ecotype "Bentheim" and the pioneer ecotype "Weerribben", respectively, was selected. Using tissue culture techniques plant material of both ecotypes was cloned in order to obtain genetically identical plants (Chapter 2). These micropropagated plants were used to set up a standardized inoculation system under axenic conditions in order to study the genetically determined nodulation ability and nitrogen-fixing capacity of Frankia strains and to select superior Frankia strains as source of inoculum (Chapter 3). The usefulness of selected Frankia strains as inoculum was further tested under more practical conditions, in perlite as model environment for nitrogen-limited conditions and in two soils, representing natural environments with different nutritional factors and different microbial populations. The results of the inoculation tests under axenic conditions were confirmed by studies under greenhouse conditions.The performance of the symbiosis was effected by many variables, e.g. the plant genotype, the Frankia strain and environmental conditions. The influence of the environmental conditions became more pronounced when plants were grown on either a sandy loam ("Bentheim") or a peat ("Weerribben") soil and inoculated with Frankia strains. Plant growth was positively influenced, e.g. by mycorrhizal fungi in "Bentheim" soil, or negatively influenced, e.g. by oomycetes in "Weerribben" soil. The effects of inoculation with Frankia on plant growth remained minimal. The establishment of the introduced Frankia strain was also dependent on the soil conditions. The introduced spore(-) Frankia strain was only able to compete with the natural spore(+) population of the "Weerribben" soil. Introduction of this strain to "Bentheim" soil did not show any establishment of the introduced strain. In contrast to the sandy loam of "Bentheim" which was rich in nutrients, the peat of "Weerribben" was a representative of poor soils. It could therefore be used for feasability studies in inoculation programmes. The use of pure cultures of Frankia as inoculum instead of soil or crushed nodules, has the advantage to prevent the contamination of the plant with root pathogens. Pure cultures did not result in a better symbiosis.Atypical Frankia strainsScreening of several isolates obtained from nodules of both alder ecotypes indicated the existence of atypical, ineffective Frankia strains. The alder clones used showed variable resistance against infection of the ineffective strains (Chapter 4). When compared with growth after the addition of a single strain dual inoculation of typical, effective Frankia strains and an ineffective Frankia strain to both alder clones showed growth increment of the plants (Chapter 5). The growth enhancing effect of the ineffective Frankia strain was not paralleled by increased number of nodules. Nothing is known yet about the growth stimulation by atypical Frankia strains. The results indicate that simultaneous inoculation of different Frankia strains to Alnus plants can be profitable for the host plant.Ribosomal RNABecause the ineffective Frankia strains lacked morphological and physiological characteristics of typical Frankia strains and because nodule formation on actinorhizal plants might be reduced or even absent, detection of the ineffective strains and studies on their competitive abilities were quite difficult. Reliable markers which could be used to detect both types of Frankia in nodules and in soil without reisolation had not been available at that moment. An attempt was made to find specific markers in a molecule which was commonly used to unravel evolutionary relationships: the 16S ribosomal RNA. New sequencing techniques allowed the rapid determination of total or almost total 16S rRNA sequences. Total 16S rRNA sequences indicated the presence of conserved and variable regions. Conserved regions had been used to investigate quantitative evolutionary relationships amoung bacteria. The conserved regions of the total 16S rRNA sequence of the effective Frankia strain Ag45/Mut15 were compared with aligned sequences of other actinomycetes and used to determine the position of the family Frankiaceae in the phylogenetic tree of the actinomycetes (Chapter 6).Analyses of variable regions of 16S rRNA of closely related organisms indicated sufficient variation, despite the fact that DNA/DNA homology studies suggested these two species might actually be one and the same. Large differences in DNA/DNA homology studies of Frankia which were also obtained between strains of one compatibility group suggested chances on large variation within the variable regions of different strains. Sequence analyses of variable regions of 16S rRNA of two ineffective Frankia strains (i.e. AgB1.9 and AgW1.1) and the effective strain Ag45/Mut1 5, all belonging to the Alnus -compatibility group, showed large differences in base composition. These sequences were used to design complementary synthetic oligonucleotides that could act as specific probes in hybridization experiments. The specificity of these probes was shown in hybridization experiments against immobilized rRNA from 23 Frankia strains belonging to different compatibiliy groups and of several related soil actinomycetes. The probes were able to distinguish between Nif +and Nif -strains, between several Nif -strains and between several Alnus compatible Nif +strains and strain AgKG'84/4 also belonging to the Alnus -compatibility group (Chapter 7). Strong strain specific sequences, however, were not obtained. The design of oligonucleotide probes opens up the possibility to investigate competitive abilities of selected strains under defined conditions, e.g. in model systems with perlite and defined Frankia strains. The question whether competition studies under these controlled conditions are ecologically relevant needs further investigations because little basic knowledge on Frankia population dynamics is yet available. The application of probes to identify introduced strains in soil remains restricted, due to the low specificity for strains. Up to now we are not able to design reliable strain specific probes that can be used to follow the establishment of introduced Frankia strains in natural environments. A much more . promising application of probes towards rRNA is concerned with the development of a genus-specific oligonucleoticle probe against Frankia (Chapter 9) that theoretically enables quantitative detection of total Frankia populations.RNA extractionThe application of oligonucleoticle probes in the detection of specific Frankia strains does not only depend on specificity of the probes but also on the development of a reliable isolation method for target sequences. Ribosomal RNA is preferable to DNA as target because of its relative abundance in large amounts in metabolically active cells. Actinorhizal nodules represent enrichments of Frankia, which are metabolically highly active and consequently contain large amounts of Frankia RNA. Our investigations resulted in the development of a rapid RNA extraction method that was sensitive enough to investigate strain composition also from very small nodules or lobes (Chapter 8). The detection of target sequences, however, remained limited by the design of specific probes and the ratios of different target sequences in one sample. For reliable signal expression in hybridization experiments quite similar amounts of target sequences per sample were needed.So far, the usefulness of rRNA sequences as targets for oligonucleoticle probes was only shown in combination with pure cultures of Frankia (Chapter 7) or in metabolically highly active enrichments, e.g. nodules (Chapter 8). Terrestrial environments like soil contain populations of many different microorganisms. These populations normally grow under suboptimal nutrition conditions. Bacteria adapt to these conditions by forming special starvation cells, which are metabolically inactive and contain only low amounts of rRNA. The starvation respons often results in viable, but non-culturable populations. The recalcitrant character of Frankia, which are difficult to isolate, makes it a useful model microorganism of soil bacteria. The application of oligonucleoticle probes for detection of Frankia in soil depends on the development of an extraction method for RNA. RNA directly isolated from soil as target for Frankia specific oligonucleoticle probes was useful in detection of Frankia (Chapter 9). Quantification of the obtained signals, however, is still unreliable because Frankia is a hyphae forming organism. It is also quite difficult to correlate cell numbers (theoretical estimation) to the amount of RNA. The concentration of these molecules in an organism is a function of the activity of the individual cell. Quantification of hybridization signals therefore depends on the availability of basic information of the metabolic activity of Frankia cells in soil. This information, however, is very difficult to obtain for recalcitrant microorganisms like Frankia. It is much easier for other microorganisms, e.g. for Streptomyces . Streptomyces spores are quite easy to isolate from soil and the establishment of Streptomyces cells, i.e. as spores or as mycelium in soil, is well studied. Quantification based on hybridization signals must be possible when this basic knowledge is available. In case of Frankia methods that enable quantification must still be developed. Similar to Streptomyces these quantification methods for Frankia can be of direct character, e.g. quantitative extraction of spores, or of indirect character, e.g. determination of mycelium by phage counts.The development of rapid and sensitive methods to detect Frankia on the basis of rRNA sequences opens up new ways to study other recalcitrant microorganisms in the environment. This molecular approach in microbial ecology can definitely further be explored when the advantages of rRNA as stable target and the rapid extraction of RNA from soil can be combined with in vitro amplification methods commonly used with DNA or mRNA. Promising approaches can also be expected in in situ studies using hybridization signal intensity of fluorescent dye labelled oligonucleotides and the amount of rRNA as criterium for bacterial activity.