| Summary: | In nature, fungal endophytes often have facultative endohyphal bacteria (FEB). Can a model plant pathogenic fungus have them, and does it affect their phenotype? We constructed a growth system/microcosm to allow an <i>F. graminearum</i> isolate to grow through natural soil and then re-isolated it on a gentamicin-containing medium, allowing endohyphal growth of bacteria while killing other bacteria. <i>F. graminearum</i> PH-1 labelled with a <i>His1mCherry</i> gene staining the fungal nuclei fluorescent red was used to confirm the re-isolation of the fungus. Most new re-isolates contained about 10 <i>16SrRNA</i> genes per fungal <i>mCherry</i> gene determined by qPCR. The <i>F. graminearum</i> + FEB holobiont isolates containing the bacteria were sub-cultured several times, and their bacterial contents were stable. Sequencing the bacterial <i>16SrRNA</i> gene from several Fg-FEB holobiont isolates revealed endophytic bacteria known to be capable of nitrogen fixation. We tested the pathogenicity of one common Fg-FEB holobiont association, <i>F. graminearum</i> + <i>Stenatrophomonas maltophilia</i>, and found increased pathogenicity. The <i>16SrRNA</i> gene load per fungal <i>His1mCherry</i> gene inside the wheat stayed the same as previously found in vitro. Finally, strong evidence was found for Fg-<i>S. maltophilia</i> symbiotic nitrogen fixation benefitting the fungus.
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