Factors Affecting Root Nodule Formation in the Frankia-actinorhizal Symbiosis
Actinorhizal plants can form symbiotic associations with root-nodule forming Gram-positive actinobacteria of the genus Frankia. These filamentous heterotrophic bacteria provide the plants with reduced nitrogen resources through nitrogen fixation. Our lab has been a pioneer in developing and using molecular tools like quantitative PCR (qPCR) to detect and quantify specific Frankia populations in soils and in root nodules. Previous studies using qPCR and Illumina sequencing suggested that root nodule formation was not a function of abundance or relative diversity of specific Frankia populations in soils. Root nodule formation could therefore be affected by alternative traits of Frankia populations present in the environment, one of which could be competition between strains in which one strain outcompetes the other for nodule formation even at low abundance. The diversity of frankiae in soils might be an additional variable since competition for nodule formation on their host plants has been described previously for different Frankia strains or populations. Vegetation has also been shown to affect the basic composition of frankiae in soils. Studies addressing these issues generally lack accurate quantification of nodulation units, i.e., numbers of individual cells and fragments potentially capable of inducing nodules on host plant species. One strain producing many small fragments with small numbers of cells could provide more nodulation units than another one developing few large colonies with comparatively more cells. The studies in this dissertation research focused on four topics involved in the establishment of the symbiosis between Frankia and the respective host plant species. The goal of the first study (chapter 2) was to investigate the potential role of infective isolates from Casuarina species on the potential establishment of noninfective Frankia strains in root nodule formation on Casuarina equisetifolia. We used quantitative PCR and in situ hybridization techniques to target strains representing specific Frankia clusters and sub-clusters to distinguish and quantify these Frankia populations in soils and root nodules of host plants in controlled microcosm studies. This study established the presence of the atypical strain R43 as a surface contaminant in the periderm of root nodules and ruled out a potential co-infection mechanism. Chapter 3 reports results of a study assessing nodule-forming capacities and competition for nodulation of two Frankia strains inoculated in defined nodulation units, i.e., specific cell or filament numbers, and identical inoculation procedure, in soil microcosms vegetated with their host plant Alnus glutinosa. Our study demonstrates that the cluster 1a Frankia strain ArI3 is much more infective and competitive for root nodule formation than cluster 1b Frankia strain Ag45/Mut15. These studies were expanded in chapter 4 to assess nodulation capacities of the prolific spore-forming Frankia strain CcI3, the type strain of Frankia casuarinae and member of cluster 1c, on its host plant Casuarina equisetifolia. Nodulation capacities were related to different nodulation units (i.e., individual cells such as spores and single vegetative cells, as well as filaments of different fragment sizes). We used qPCR and fluorescence microscopy to quantify the nodulation units and the role of nodulation units in estimating the efficiency of Frankia inoculum in forming root nodules. Finally, chapter 5 reports on studies assessing host plant effects on the abundance and distribution of introduced or indigenous Frankia populations in soils and in root nodules. Host plants belonging to different Alnus species were used in microcosm setups with silty clay loam from Illinois with indigenous Frankia populations, and sandy soils from Bastrop, Texas, with introduced Frankia strains belonging to clusters 1a, 1b, 1c, 3 and 4 to study vegetation effects. This study showed large differences between indigenous Frankia populations detected in soils and in root nodules of different host plants, thereby confirming that plant species are selecting specific Frankia populations from soil for root nodule formation independent of the abundance or relative diversity of specific Frankia populations in soils. Introducing Frankia strains into sandy soils showed a competitive advantage of Frankia cluster 1a strain ArI3 over other strains in forming root nodules, however, this strain is outcompeted by other strains during saprotrophic growth in soil. This competitive advantage of cluster 1a strain has been reported in chapter 2 as well, however, is only observed when strains were introduced into soils, and not in established populations in soils. This effect could therefore be related to the inoculation of the strain, and thus reflect an artifact that is not prevalent in indigenous Frankia populations where plant selection might play a major role in root nodule formation by specific Frankia populations. This potential competition for nodule formation in introduced Frankia populations is important and needs to be investigated in field studies to improve Frankia inoculum for use in agroforestry. Studies on the presence and importance of multiple strains in single root nodules is generally lacking and hence future studies should be focused on the role of atypical strains isolated from one host plant but found to be infective on other host plants as well as on strains of cluster 4 Frankia, which represent atypical and generally non-nitrogen fixing frankiae.
Frankia, competition, root nodule, quantification, host plant
Vemulapally, S. (2021). Factors affecting root nodule formation in the Frankia-actinorhizal symbiosis (Unpublished dissertation). Texas State University, San Marcos, Texas.