Endohyphal bacterial communities modulate fungal responses to hyphosphere-associated bacteria in Fusarium

Fungi and bacteria form complex associations in soil environments, which can impact their symbiotic relationships. While previous studies have examined either endohyphal bacteria or hyphosphere-associated bacterial community interactions with fungi independently, a critical knowledge gap remains related to how hyphosphere-associated bacteria modulate the endohyphal bacterial communities and fungal responses. This study investigated the impact of hyphosphere-associated bacteria co-culture with three Fusarium isolates containing natural endohyphal bacteria (composed primarily of Cutibacterium , Acinetobacter , Pelomonas , Achromobacter , Citrobacter, and other less abundant species) and their antibiotic-treated counterparts with reduced endohyphal bacterial communities. The study determined the changes in fungal growth and microbiome composition when co-cultured with individual or a bacterial mix of four rhizosphere bacterial isolates ( Pseudomonas , Bacillus , Stenotrophomonas , and Rhizobium ). Results showed that endohyphal bacteria modulated fungal responses to hyphosphere-associated bacteria co-culture, with wild-type fungi typically exhibiting lower growth inhibition than their counterparts with reduced diversity and quantity of endohyphal bacteria. However, when the fungi were exposed to a synthetic hyphosphere-associated bacterial mix, all fungal isolates stimulated the endohyphal bacterial community leading to higher endohyphal bacterial diversity compared to single-species hyphosphere-associated bacterial treatments. These findings highlight the importance of considering complex interactions within the fungal microbiome, demonstrating that endohyphal bacteria respond dynamically to the composition of hyphosphere-associated bacterial communities. Our results also provide new insights into how the interplay between endohyphal and hyphosphere-associated bacterial communities shape fungal biology in soil environments.