A host-derived chimeric peptide protects citrus against Huanglongbing without threatening the native microbial community of the phyllosphere

Abstract

Introduction: The application of host-derived antibacterial peptides has been highlighted as a potential efficacious and safe tool for the treatment of Huanglongbing (HLB), the most devastating disease of citrus. However, pathogenic bacteria such as HLB-causing Candidatus liberibacter asiaticus (CLas) often develop resistance against the host antibacterial peptides. We showed that chimeras containing two different host antibacterial peptides not only retain antibacterial activity but also overcome bacterial resistance and enhance plant defence responses. Also, chimeric peptides can have an off-target impact on the structure and function of plant-associated microbiomes. However, there is a lack of understanding of the impact of the chimeric peptide therapy on the microbial structure in the citrus phyllosphere while reducing the CLas titre. Here, we aim to evaluate the efficacy of a chimeric peptide (UGK17) to reduce CLas titre, inducing plant defence response and impacting the microbiome associated with the citrus phyllosphere. Material and Method: Leaf samples were collected from orange and grapefruit trees in Texas and identified as old and young leaves according to their maturity. We collected three different types of leaves based on their infection and symptoms: healthy, symptomatic (infected with typical symptoms), and asymptomatic (infected without symptoms). In planta assay was performed by dipping the leaves in the 0, 5 and 25 μM of UGK17 solutions for 48 h. The quantifications of CLas titre and pathogenesis-related (PR) gene expression were done by quantitative polymerase chain reaction (qPCR) and reverse transcription-qPCR, respectively. Amplicon sequencing was done to evaluate the impact of UGK17 on individual bacterial community structures. In addition, we performed an ex planta assay to assess the effect of UGK17 on the growth of bacterial isolates including Liberibacter crescens instead of unculturable CLas predominant in the phyllosphere. Result: The UGK17 treatment reduced the CLas titre in both asymptomatic and symptomatic citrus leaves, regardless of the age of the leaves. The UGK17 application augmented the PR gene expression. In ex planta assay, the growth of L. crescens along with four other strains belonging to the family Rhizobiaceae, was significantly inhibited by the UGK17 while the growth of 74 strains were unaffected. Additionally, there was no statistically significant changes in the microbial community structure with UGK17 treatment. Conclusion: Our results suggest that the chimeric peptide therapy is a promising solution to combat HLB by targeting mainly Gram-negative pathogens and enhancing the plant immune responses without impairing the indigenous microbial community in the citrus phyllosphere.