Characterization of Endofungal Bacteria and Their Role in the Ectomycorrhizal Fungus Helvella bachu

Helvella bachu, an ectomycorrhizal fungus, forms a symbiotic relationship with Populus euphratica, a rare and endangered species crucial to desert riparian ecosystems. In this study, endofungal bacteria (EFBs) within the fruiting bodies of H. bachu were confirmed by a polyphasic approach, including genomic sequencing, real-time quantitative PCR targeting the 16S rRNA gene, full-length and next-generation sequencing (NGS) of the 16S rRNA gene, and culture methods. The genera Stenotrophomonas, Variovorax, Acidovorax, and Pedobacter were abundant in the EFBs of fruiting bodies associated with three Populus hosts and were consistently present across different developmental stages. Notably, S. maltophilia and V. paradoxus were detected in high abundance, as revealed by full-length 16S rRNA sequencing, with S. maltophilia also isolated by culture methods. KO-pathway analysis indicated that pathways related to primary, secondary, and energy metabolism were predominantly enriched, suggesting these bacteria may promote H. bachu growth by producing essential compounds, including sugars, proteins, and vitamins, and secondary metabolites. This study confirmed the presence of EFBs in H. bachu and provided the first comprehensive overview of their structure, functional potential, and dynamic changes throughout fruiting body maturation, offering valuable insights for advancing the artificial domestication of this species.