Shifts in bacterial community composition during symbiotic seed germination of a terrestrial orchid and effects on protocorm development.

Fungi and bacteria often occupy very similar niches; they interact closely with each other, and bacteria can provide direct or indirect benefits to plants that form mutualistic interactions with fungi. In orchids, successful seed germination largely depends on compatible mycorrhizal fungi, but whether and how bacteria contribute to seed germination and protocorm development remains largely unknown. Here, we performed field and laboratory experiments to assess the potential role of bacteria in mediating seed germination and protocorm development in the terrestrial orchid Gymnadenia conopsea. Our results suggested that bacterial and fungal communities differ between developmental stages in the germination process. The diversity of bacterial and fungal communities and their interaction network in germinating seeds (Stage 1) differed significantly from those in later developmental stages (Stages 2-5). Pseudomonas gradually became the dominant bacterial group as the protocorms matured and showed a positive association with Ceratobasidiaceae fungi. Seed germination tests in vitro demonstrated that co-inoculation of Ceratobasidium sp. GS2 with Pseudomonas isolates significantly improved protocorm growth and development, suggesting that the observed increase in Pseudomonas abundance during protocorm development directly or indirectly improves the growth of germinating seeds. Overall, our findings indicate that bacteria may exert non-negligible effects on seed germination of orchids and, therefore, offer valuable perspectives for future strategies for conservation and cultivating orchid species.

Importance: It is well known that orchid seeds depend on mycorrhizal fungi to supply the necessary nutrients that support germination in natural environments. Apart from fungi, bacteria may also be involved in the germination process of orchid seeds, but so far, their role has not been intensively studied. This research provides evidence that bacterial community composition changes during seed germination of the terrestrial orchid Gymnadenia conopsea. Interestingly, in vitro experiments showed that Pseudomonas spp., which were the most dominant bacteria in the later germination stages, improved protocorm growth. These results suggest that bacteria contribute to the germination of orchid seeds, which may open new perspectives to apply bacteria as a biofertilizer in the introduction and restoration of G. conopsea populations.