Control efficiency and Huanglongbing resistance-related clues mediated by novel Hermetia illucens frass formulation

The preventive and curative management of citrus Huanglongbing (HLB) remains a major global challenge in citrus production. As an emerging microbial fertilizer rich in antimicrobial peptide mixtures, Hermetia illucens frass (HIF) exhibits promising antibacterial potential against HLB-associated pathogens. In this study, we evaluated the suppressive effects of HIF on Candidatus Liberibacter asiaticus (CLas) infection and explored the underlying physiological, metabolic, and multi-omic regulatory mechanisms in CLas-infected citrus nursery trees grown in pots. Quantitative real-time PCR analysis showed that 20 and 25 consecutive soil applications of HIF significantly reduced CLas titers by 52.61% and 61.85%, respectively, and citrus leaves with typical chlorotic and mottled symptoms gradually recovered normal green coloration after 20 rounds of treatment. Phytohormone profiling indicated that the endogenous contents of auxin, cytokinin, and salicylic acid were significantly upregulated following HIF application. Non-targeted metabolomics further revealed that HIF treatment markedly increased the accumulation of 375 and 724 differential metabolites in citrus tissues, including ketones, aldehydes, terpenoids, flavonoids, alkaloids, coumarins, steroids, and polyphenols. Transcriptomic and metabolomic analyses identified 9 significantly upregulated KEGG pathways in leaves and 26 in roots after HIF treatment. Integrated multi-omic comparisons yielded 15 co-upregulated pathways from transcriptome-metabolome pairing, 4 from transcriptome-proteome pairing, and 1 from proteome-metabolome pairing. Notably, α-linolenic acid metabolism was consistently activated across transcriptomic, proteomic, and metabolomic datasets, representing a core conserved signaling pathway responding to HIF treatment. Microbial community analysis characterized the top 10 dominant bacterial genera in both HIF material and HIF-treated citrus tissues. Furthermore, HIF contained abundant antimicrobial secondary metabolites, such as lipids, benzenoids, polyketides, and phenylpropanoids. HPLC detection confirmed the presence of the lipopeptides surfactin and iturin, and metagenomic alignment predicted a total of 467 antimicrobial peptides classified as attacin-like, defensin-like, and cecropin-like peptides. Collectively, these phenotypic, physiological, and multi-omic results provide multi-layered validation clues for research on utilizing HLF to combat Huanglongbing.