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.