Biochar-associated persistent free radicals inhibit wheat growth: novel insights into ecoenzymatic stoichiometry.

Biochar-associated persistent free radicals (PFRs) can directly induce oxidative damage in plants. However, this toxic mechanism may not be fully applicable to plants growing in a real soil ecosystem considering the complexity of the soil environment. In this study, biochar was applied to soil at rates ranging from 0 to 20 g kg-1, and wheat growth was inhibited at 20 g kg-1, but did not at 1-10 g kg-1. Notably, inhibitor and sterilization experiments indicated that PFRs and their derived hydroxyl radical (˙OH) were the primary factors inhibiting wheat growth, and wheat damage degree primarily depended on the impact of free radicals on rhizosphere microbes rather than direct damage to wheat itself. Integrated analyses of enzymatic stoichiometry, microbial community structure, and transcriptomic profiles analysis further revealed that at 20 g kg-1 dosage, PFRs and ˙OH aggravated microbial C and P limitations, prompting rhizosphere microbes to preferentially allocate metabolic energy for alleviating nutrient limitations. This reduces the abundance of disease-suppressive bacteria (e.g., Actinobacteria, Gemmatimonadetes, and Nitrospirae) and bacterial α-diversity, ultimately increasing wheat susceptibility to free radical stress. By contrast, free radicals did not induce stoichiometric imbalance at 1-10 g kg-1 dosage. Rhizosphere microbes could allocate more energy to activate the wheat defense system, including salicylic acid metabolism, L-phenylalanine metabolism, and the MAPK signaling pathway, thereby reducing free radical-induced damage to wheat. These findings highlight a previously overlooked mechanism of free radical-induced phytotoxicity, providing a foundation for developing improved biochar application strategies.