Unveiling root nodulation in Tribulus terrestris and Roystonea regia via metagenomics analysis

Root nodule symbiosis is traditionally recognized in the Fabales, Fagales, Cucurbitales, and Rosales orders within the Rosid I clade of angiosperms. However, ambiguous root nodule formation has been reported in Zygophyllaceae and Roystonea regia (Arecaceae), although a detailed analysis has yet to be conducted. We aimed to perform morphological analyses of root structures in these plants and utilize metagenomic techniques to identify and characterize the bacterial populations within the nodule-like structures. We collected root samples of Tribulus terrestris (Zygophyllaceae) and Roystonea regia from West Asia and the Caribbean, respectively. We conducted detailed morphological analyses of nodule-like structures, isolated and genome-sequenced the endophytes, and employed metagenomic techniques to identify the bacterial populations within these formations. We observed nodule-like structures in both plant species. Symbiosomes, which are hallmark structures of nodulating plants, were not detected. Metagenome sequence data analysis revealed potential nodulating and nitrogen-fixing bacteria in the nodule-like structures of both species. Canonical nodulation and nitrogen-fixation genes were identified in microbes associated with the nodules. However, the phylogenomic analysis showed that the bacteria isolated from T. terrestris and R. regia are within Gammaproteobacteria and Bacilli, which are not typically known as nodulating bacteria. The observed structures differ significantly from traditional nodules found in legumes and actinorhizal plants, suggesting unique characteristics with hosting nitrogen-fixing bacteria. Although bacteria identified through in silico analysis or culture are well-known nitrogen-fixers, their specific role in root nodule formation remains to be investigated.