| Summary: | Exploration of the effect of soil bacteria on growth and metabolism of beneficial root endophytic fungi is relevant to promote favorable associations between microorganisms of the plant rhizosphere. Hence, the interaction between the plant-growth-promoting fungus <i>Piriformospora indica</i> and different soil bacteria was investigated. The parameters studied were fungal growth and its amino acid composition during the interaction. Fungus and bacteria were confronted in dual cultures in Petri dishes, either through agar or separated by a Perspex wall that only allowed the bacterial volatiles to be effective. Fungal growth was stimulated by <i>Azotobacter chroococcum</i>, whereas <i>Streptomyces anulatus</i> AcH 1003 inhibited it and <i>Streptomyces</i> sp. Nov AcH 505 had no effect. To analyze amino acid concentration data, targeted metabolomics was implemented under supervised analysis according to fungal-bacteria interaction and time. Orthogonal partial least squares-discriminant analysis (OPLS-DA) model clearly discriminated <i>P. indica</i>−<i>A. chroococcum</i> and <i>P. indica</i>−<i>S. anulatus</i> interactions, according to the respective score plot in comparison to the control. The most observable responses were in the glutamine and alanine size groups: While <i>Streptomyces</i> AcH 1003 increased the amount of glutamine, <i>A. chroococcum</i> decreased it. The fungal growth and the increase of alanine content might be associated with the assimilation of nitrogen in the presence of glucose as a carbon source. The <i>N</i>-fixing bacterium <i>A. chroococcum</i> should stimulate fungal amino acid metabolism via glutamine synthetase-glutamate synthase (GS-GOGAT). The data pointed to a stimulated glycolytic activity in the fungus observed by the accumulation of alanine, possibly via alanine aminotransferase. The responses toward the growth-inhibiting <i>Streptomyces</i> AcH 1003 suggest an (oxidative) stress response of the fungus.
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