Foliar Epichloë gansuensis Endophyte and Root-Originated Bacillus subtilis LZU7 Increases Biomass Accumulation and Synergistically Improve Nitrogen Fixation in Achnatherum inebrians

Although drunken horse grass (<i>Achnatherum inebrians</i>) can be simultaneously infected by the foliar endophyte <i>Epichloë gansuensis</i> and colonized by <i>Bacillus subtilis</i>, it remains unclear whether <i>Epichloë</i> endophyte symbiosis influences <i>B. subtilis</i> colonization, as well as how their interaction affects nitrogen fixation and assimilation. The purpose of the present study was to investigate whether <i>E. gansuensis</i> endophyte infection facilitates the colonization of <i>B. subtilis</i> in the roots of host plants, with a focus on understanding the interaction effects of the <i>E. gansuensis</i> endophyte and <i>B. subtilis</i> on plant growth and nutrient absorption. In this study, we measured the colony growth rate of <i>B. subtilis</i> LZU7 when co-cultured with <i>E. gansuensis</i> strains. In addition to an in vitro test, we investigated the root colonization of <i>Epichloë</i> endophyte-infected plants (E+) and <i>Epichloë</i> endophyte-free plants (E-) with the GFP-tagged <i>B. subtilis</i> LZU7 in an inoculation test. Furthermore, we evaluated the interactions between <i>E. gansuensis</i> endophyte symbiosis and <i>B. subtilis</i> LZU7 colonization on the dry weight, nitrogen fixation, nitrogen converting-enzyme activity, and nutrients for E+ and E- plants by labeling with ¹⁵N₂. The results showed that the growth rates of <i>B. subtilis</i> LZU7 were altered and increased in a co-culture with the <i>E. gansuensis</i> endophyte. A significantly greater colonization of GFP-tagged <i>B. subtilis</i> LZU7 was detected in the roots of E+ plants compared with the roots of E- plants, suggesting that <i>E. gansuensis</i> endophyte symbiosis enhances the colonization of beneficial microorganisms. The combination of <i>E. gansuensis</i> endophyte symbiosis and <i>B. subtilis</i> LZU7 inoculation significantly altered the expression of the nitrogenase (<i>nifH</i>) gene, thereby promoting increased biological nitrogen fixation (BNF). The <i>E. gansuensis</i> endophyte infection and inoculation with <i>B. subtilis</i> LZU7 significantly increased δ15NAir in plants. Co-inoculation with the <i>E. gansuensis</i> endophyte and <i>B. subtilis</i> LZU7 significantly elevated NH₄⁺ accumulation in the roots, depleted the NH₄⁺ availability in the surrounding soil, and showed no measurable impact on the foliar NH₄⁺ content. The observed alterations in the NH₄⁺ content were linked to nitrogen-fixing microorganisms that promoted nitrogen fixation, thereby enhancing nitrogen uptake and contributing to greater biomass production in <i>A. inebrians</i>. Our findings highlighted the fact that a foliar symbiosis with the <i>E. gansuensis</i> endophyte enhances the recruitment of beneficial bacteria, and that the resulting interaction significantly impacts nitrogen fixation, assimilation, and allocation in host plants.