Keystone taxa of phyllosphere microbiome confer resistance to citrus bacterial canker in pomelo via multiple mechanisms

Citrus bacterial canker (CBC) is a globally important citrus disease caused by Xanthomonas citri subsp. citri (Xcc). Increasing evidence shows that the plant microbiome is crucial for host growth performance and health maintenance, among which the keystone taxa stand out due to their indispensable roles in microbiome homeostasis. However, how keystone taxa in the phyllosphere microbiome contribute to disease resistance remains unclear. In this study, we characterized the phyllosphere bacterial community of pomelo across an annual cycle and found that amino acids in leaves were the main drivers of the bacterial community structure. Meanwhile, five OTUs were identified as keystone taxa. A total of 587 phyllosphere bacterial strains were isolated, among which six strains belonging to Methylobacterium, Sphingomonas, Massilia, and Paenibacillus were identified as the corresponding keystone strains. We further constructed a consortium with these six strains to test its role in controlling CBC in planta. Network analysis reveals that consortium inoculation increased the phyllosphere bacterial community stability, whereas Xcc inoculation decreased it. However, dual inoculation of the consortium and Xcc restored community stability compared to the control. Interestingly, the inoculated keystone strains, if not all, still appeared as keystone taxa in the microbiomes of the control, consortium inoculation, and dual inoculation treatments, but not in the Xcc-inoculated treatment. Moreover, the consortium inoculation significantly increased the defense-related enzyme activities such as PPO, POD, and PAL in leaves, suggesting a triggered plant immune response. In vitro assays indicated that these keystone strains showed either antagonistic activity against Xcc or siderophore-producing activity. Finally, the consortium inoculation significantly reduced the disease index by 78% in planta. Taken together, these results suggest that the keystone taxa of the phyllosphere microbiome can confer disease resistance to the host via multiple mechanisms, especially by maintaining phyllosphere microbiome homeostasis.