Morphogenesis and metabolomics reveal the compatible relationship among Suillus bovinus, Phialocephala fortinii, and their co-host, Pinus massoniana

ABSTRACT Ectomycorrhizal (ECM) fungi and dark septate endophytes (DSEs) can both form a symbiotic relationship with the same host plant. However, the interactions that occur among these two types of fungi and their co-hosts are largely unknown. Here, we investigated interactions that occur among the ECM fungus Suillus bovinus, the DSE Phialocephala fortinii, and their co-host Pinus massoniana. We used both scanning electron microscopy and optical microscopy to characterize the morphogenesis of the two symbionts and employed the ultra-high-performance liquid chromatography–tandem mass spectrometry technique to assess the effects of fungal inoculation on the root metabolome. Under pure culture conditions, no synergistic or antagonistic effects were observed between Phi. fortinii and S. bovinus. Generally, S. bovinus and Phi. fortinii can simultaneously colonize P. massoniana roots without affecting each other’s symbiotic processes. S. bovinus can colonize the root locus where Phi. fortinii has already invaded but not vice versa, which may be due to the physical barrier effect of the mantle. Both fungi can significantly promote the growth of P. massoniana, and they have a synergistic effect on host N and K uptake. Metabolite accumulation patterns in roots inoculated with Phi. fortinii and/or S. bovinus were greatly altered, especially with respect to organic acids, flavonoids, lipids, and phenolic acids. S. bovinus inoculation significantly enhanced root flavonoid biosynthesis, whereas Phi. fortinii and dual-inoculation treatments mainly induced phenylpropanoid biosynthesis. These findings reveal compatible relationships among P. massoniana, S. bovinus, and Phi. fortinii, and suggest a theoretical basis for ECM fungi and DSE co-application when cultivating seedlings. IMPORTANCE The prevalence of both ectomycorrhizal fungi and dark septate endophytes in the roots of a wide spectrum of tree species is well recognized. In this study, we investigated the interactions that occur among the ECM fungus S. bovinus, the DSE Phi. fortinii, and their co-host, P. massoniana. The two fungi can simultaneously colonize P. massoniana roots without affecting each other’s symbiotic processes. S. bovinus appears to be superior to Phi. fortinii in microniche competition, which may be due to the physical barrier effect of the mantle. The two fungi have different effects on root metabolite accumulation patterns. S. bovinus inoculation significantly enhanced root flavonoid biosynthesis, whereas Phi. fortinii and dual-inoculation treatments mainly induced phenylpropanoid biosynthesis. This is the first study revealing the morphological and metabolic mechanisms that contribute to the compatible relationship among ECM fungi, DSEs, and their co-host.