Citrus spp. represent an economically important fruit tree crop worldwide. However, molecular mechanisms underlying the interaction between citrus and the Colletotrichum gloeosporioides remain largely unexplored. In this study, we analyzed the physiological and transcriptomic changes in Citrus sinensis at different stages of incubation with C. gloeosporioides. The results indicated that C. gloeosporioides infection rapidly triggered necrosis in the epicarp of C. sinensis fruits, decreased the total flavonoid contents, and suppressed the activity of catalase, peroxidase, and superoxide dismutase enzymes. Upon inoculation with C. gloeosporioides, there were 4600 differentially expressed genes (DEGs) with 1754 down-regulated and 2846 up-regulated after six hours, while there were only 580 DEGs with 185 down-regulated and 395 up-regulated between six and twelve-hours post-inoculation. Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes enrichment analysis indicated that the DEGs, which exhibited consistent up-regulation, were associated with metabolic processes and stress responses. Through Weighted Gene Co-Expression Network Analysis, 11 key genes have been identified that could potentially play a role in the transcriptional regulation of this process, including the transcription factor bHLH189. Furthermore, the infection of C. gloeosporioides had a notable effect on both the flavonoid metabolism and the metabolic pathways related to reactive oxygen species. Our findings help to understand the interaction between citrus and C. gloeosporioides and unveil how new insights into how C. gloeosporioides circumvents citrus defense mechanisms.

HLB is a severe and devastating disease affecting citrus plants, for which no cure is currently available. The pathogen CLas is an unculturable, phloem-limited α-proteobacterium associated with HLB. Phages, known for their ability to directly lyse pathogenic bacteria, have been widely recognized as effective control agents in both medical and agricultural fields. In this study, we identified 191 prophages across 48 Liberibacter genomes using comparative genomics and phylogenetic methods. These prophages were classified into 17 distinct types, with 13 novel types, named NF1 to NF13. Diversity analysis revealed that the number of prophages per genome ranged from one to eight, with an average of four. Annotation showed that the genome sizes of NF1–NF13 ranged from 10,160 bp to 72,736 bp, with an average size of 27,616 bp, containing between 7 and 66 genes (average: 27 genes). Further functional enrichment of the sequences within 5 kb upstream and downstream of the integration site identified six main aspects, involving four core biological processes: genome structure modification, gene expression regulation, stress response activation, and metabolic pathway modulation. It is speculated that the insertion of the prophage significantly impacts the host. Six virulence factors, including carB, clpC, flhA, algW, rffG, and IlpA, were identified in the prophages. Interestingly, prophages containing carB or clpC are predominantly found in CLas, which may contribute to the global colonization of CLas. Notably, among the 35 identified Type 4 prophages, 19 were found to be active. Type 4 is the only active prophage type, making it a promising candidate for developing phage therapy against CLas. This study is significant for a deeper understanding of the diversity of Liberibacter and the interactions between prophages and their hosts.

Citrus bacterial canker (CBC), caused by Xanthomonas citri subsp. citri (Xcc), threatens citrus production worldwide. Long-term dependence on copper-based bactericides not only poses environmental risks but also accelerates the emergence of copper-resistant Xcc strains. To develop safe and efficient alternative control strategies, 72 bacterial strains were isolated from the phyllosphere of citrus plants naturally infected by CBC and identified by 16S rRNA sequencing. Using an Xcc-GFP-based screening method, we systematically screened a highly effective strain, which was identified as Bacillus velezensis RF2 (Bv-RF2). Both inhibition zone assays and bioactivity tests of the crude methanolic extract of Bv-RF2 demonstrated stable antibacterial activity under UV irradiation, protease treatment, high temperature, and across a wide pH range. Whole-genome sequencing and antiSMASH analysis revealed multiple predicted NRPS/PKS-type biosynthetic gene clusters (BGCs). Together with metabolomic profiling, these data provide hypotheses for candidate metabolites that may contribute to antagonism. Bv-RF2 was associated with the induction of PR gene expression in immune-related pathways implicated in CBC responses. In sweet orange leaves, Bv-RF2 infiltration was associated with transient induction of defense-related (PR) genes, consistent with an ISR-like, priming-related response. In addition, Bv-RF2 inhibited the growth of fungal pathogens associated with citrus anthracnose and brown spot in vitro, indicating broad inhibitory potential under the tested conditions. Collectively, Bv-RF2 represents a promising candidate for developing environmentally friendly strategies against CBC and other citrus diseases.