Abstract In nature, fungal endophytes often have facultative endohyphal bacteria. Can a fungal pathogen such as Fusarium graminearum , pathogenic on wheat, get facultative endohyphal bacteria from soil (FEB) and how does the FEB affect the fungal phenotype? We constructed a growth system/microcosm that allowed a molecularly well-studied F. graminearum isolate, PH-1, to grow through natural soil and then be re-isolated on a gentamicin-containing medium, allowing endohyphal growth of the bacteria while killing eventual bacteria growing on the agar medium. We had labelled the F. graminearum PH-1 with a His1mCherry gene staining the fungal nuclei fluorescent red to confirm re-isolation of the same isolate we sent through the soil. Through qPCR of the 16SrRNA gene in the bacteria using universal primers combined with qPCR of the mCherry gene of DNA from the re-isolated cultures of the Fg-FEB holobionts growing on gentamicin-containing media, it was found that most of the holobiont isolates contained about 10 16SrRNA genes per fungal mCherry gene. The Fg-FEB holobiont isolates were sub-cultured several times, and the FEB content on lab media was stable. Sequencing the 16SrRNA gene from several Fg-FEB holobiont isolates revealed known endophytic bacteria capable of nitrogen fixation. We compared the pathogenicity of one of the Fg-FEB holobionts Fg-S.maltophilia , with the background without FEB and found that it was more pathogenic than without FEB. We could also show that the bacterial 16SrRNA load per fungal His1mCherry gene inside the wheat stayed the same as in culture. Finally, we tested if the Fg-S.maltophilia was capable of nitrogen fixation and could show that it, on a nitrogen-free medium, formed a dense mycelium containing proteins at similar levels as on regular nitrogen-containing media. Our results could indicate that naturally occurring fungal pathogens outside lab conditions might contain facultative endohyphal bacteria, positively affecting their pathogenicity and ecological fitness.

The wheat head blight pathogen <i>Fusarium graminearum</i> recruits facultative endohyphal bacteria from the soil, making the fungal-bacterial holobiont nitrogen-fixing and increasing the fungal pathogenicity

The wheat head blight pathogen Fusarium graminearum recruits facultative endohyphal bacteria from the soil, making the fungal-bacterial holobiont nitrogen-fixing and increasing the fungal pathogenicity