A synthetic microbial community derived from healthy apple rhizosphere alleviates apple replant disease

Apple replant disease (ARD) poses a major threat to global orchard productivity, yet its biological causes remain poorly understood. Although microbial dysbiosis in replant soils has been recognized as a major contributing factor, the specific pathogenic agents involved and the efficacy of synthetic microbial communities in mitigating ARD remain unclear. In this study, we integrated physiological, transcriptomic, metabolomic, and microbiome analyses to investigate the effects of replant soils on the growth of <i>Malus domestica</i> rootstock M26. Absolute quantification amplicon sequencing of 16S rRNA and ITS regions revealed a marked decline in rhizospheric microbial diversity in replant soils compared to fallow controls, accompanied by an enrichment of fungal genera such as <i>Fusarium</i>, <i>Aspergillus</i>, and <i>Acremonium</i>. Pathogenicity assays and seedling colonization experiments verified strong pathogenicity for five isolates-<i>Acremonium</i> sp., <i>Aspergillus niger</i>, <i>Fusarium solani</i>, <i>Macrophomina phaseolina</i>, and <i>Aspergillus stellatus</i>-implicating them as potential causal agents of ARD. High-throughput culturing and confrontation assays were used to isolate and screen candidate microbial antagonists. A synthetic microbiota (SynMs) composed of 12 bacterial strains and <i>Trichoderma</i> sp. was developed. Inoculation with SynMs significantly inproved plant height by 133% (<i>P</i> < 0.05) and total root length by 186% (<i>P</i> < 0.01), and effectively suppressed pathogen proliferation of the five pathogenic isolates in replant soils. Collectively, these findings identify key fungal pathogens underlying ARD and propose a sustainable microbiota-based strategy for its effective mitigation, offering both mechanistic insights and practical solutions for microbiome-informed orchard management.