Trichoderma afroharzianum T22 Induces Rhizobia and Flavonoid‐Driven Symbiosis to Promote Tolerance to Alkaline Stress in Garden Pea

Soil alkalinity is a limiting factor for crops, yet the role of beneficial fungi in mitigating this abiotic stress in garden pea is understudied. In this study, Trichoderma afroharzianum T22 colonised the roots of garden pea cultivars exposed to soil alkalinity in a host-specific manner. In alkaline-exposed Sugar Snap, T22 improved growth parameters, consistent with increased tissue mineral content, particularly Fe and Mn, as well as enhanced rhizosphere siderophore levels. The split-root assay demonstrated that the beneficial effects of T22 on alkaline stress mitigation are the result of a whole-plant association rather than localised root-specific effects. RNA-seq analysis showed 575 and 818 differentially expressed genes upregulated and downregulated in the roots inoculated with T22 under alkaline conditions. 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 homoeostasis. Further, a flavonoid precursor restored plant health even in the absence of T22, confirming the role of microbial symbiosis in mitigating alkaline stress. 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 soil alkalinity. 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 mineral mobilisation, while T22-mediated alkaline stress 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 alkaline media. Thus, the beneficial effect of T22 on rhizobia likely stems from their interactions, not solely from the improved mineral status, particularly Fe, in plants. This study provides the first mechanistic insights into T22 interactions with host and rhizobia, advancing microbiome strategies to alleviate soil alkalinity in peas and other legumes.