Trichoderma afroharzianum T22 induces rhizobia and flavonoid-driven symbiosis to promote iron deficiency tolerance in garden pea

ABSTRACT Iron (Fe) deficiency is a limiting factor for legumes, yet the role of beneficial fungi in mitigating Fe deficiency in garden pea is understudied. In this study, Trichoderma afroharzianum T22 colonized the roots of garden pea cultivars exposed to alkali-mediated Fe-deficiency in a host- specific manner. In Fe-deficient Sugar Snap, T22 improved growth parameters consistent with increased tissue Fe and rhizosphere siderophore. The split-root assay demonstrated that the beneficial effects of T22 on Fe deficiency mitigation are the result of a whole-plant association rather than localized root-specific effects. RNA-seq analysis showed 575 and 818 differentially expressed genes upregulated and downregulated in the roots inoculated with T22 under Fe deficiency. The upregulated genes were mostly involved in the flavonoid biosynthetic pathway (monooxygenase activity, ammonia-lyase activity, 4-coumarate-CoA ligase), along with genes related to mineral transport and redox homeostasis. Further, a flavonoid precursor restored plant health even in the absence of T22, confirming the role of microbial symbiosis in mitigating Fe deficiency. Interestingly, T22 restored the abundance of rhizobia, particularly Rhizobium leguminosarum and Rhizobium indicum , along with the induction of NifA , NifD , and NifH in nodules, suggesting a connection between T22 and rhizobia under Fe-starvation. Further, the elevated rhizosphere siderophore, root flavonoid, expression of PsCoA (4-coumarate-CoA ligase) as well as the relative abundance of TaAOX1 and R. leguminosarum diminished when T22 was substituted with exogenous Fe. This suggests that exogenous Fe eliminates the need for microbiome-driven Fe mobilization, while T22-mediated Fe mitigation depends on flavonoid- driven symbiosis and R. leguminosarum abundance. It was further supported by the positive interaction of T22 on R. leguminosarum growth in Fe-deficient media. Thus, the beneficial effect of T22 on rhizobia likely stems from their interactions, not just improved Fe status in plants. This study provides the first mechanistic insights into T22 interactions with host and rhizobia, advancing microbiome strategies to alleviate Fe deficiency in peas and other legumes.