First evidence of presence and role of endofungal bacteria (EFB) in orchid mycorrhizal fungi (OMF)

Plant-microbial interactions, especially with fungi and bacteria, are essential for plant existence. Yet, many symbiotic interactions remain undiscovered. Orchids, one of the largest angiosperm families, depend on orchid mycorrhizal fungi (OMF). In the study of orchids, the focus has primarily been on OMF. No studies have documented whether OMF hosts endofungal bacteria (EFB), and what growth-promoting benefits these EFB might confer. We discovered and documented EFB within OMF using molecular, fluorescent (FISH), and electron (SEM) microscopy techniques, isolated EFB, and empirically tested their role in orchid seed germination and protocorm development. We isolated and identified 9 unique OMF-EFB pairs from the root tissues and protocorms of Platanthera chapmanii. Two of them were obtained from protocorms and seven from roots. In the germination tests, germination percentages after 120 days were highest (66.02%) when OMF containing their EFB were used. When OMF isolates lacking the endosymbiont EFB were utilized, the germination percentages were lower (54.74%). Notably, the EFB alone also improved germination (51.35%) compared to the uninoculated seeds (33.42%). This study marks the first documentation of the presence of EFB within OMF. And the functional role of these bacteria as mycorrhizal helper bacteria or as plant growth-promoting bacteria during orchid seed germination.