Response Of Citrus And Citrus Phyllosphere Microbiome To Infection Of Diaporthe Citri

Melanose disease caused by Diaporthe citri is one of the most widely prevalent diseases of citrus.Previous studies indicated that citrus host can activate a series of defense responses after D.citri infection,including plant cell division and coumarin generation.However,direct evidence of gene regulation for this defense process is lacking.Here,we used RNA-Seq to profile the regulation pattern of citrus leaves in response to D.citri infection.Moreover,plant and their microbiome collectively form a holobiont.In contrast to the intensively studied roles of the rhizosphere microbiome in suppressing plant pathogens,the collective community‐level change and effect of the phyllosphere microbiome in response to pathogen invasion remains largely elusive.Based on the characteristics of defense response in citrus,we investigate the changes in phyllosphere microbiome between infected and uninfected citrus leaves by D.citri.We further examine the antagonistic activity of the bacteria associated with the microbiome change by in vitro dual culture assays and glasshouse experiments.1.The regulation pattern of citrus leaves in response to D.citri infectionThe differentially expressed genes were computed by RNA-Seq analysis between D.citri-versus mock-inoculated samples at 3 and 14 days post inoculation,representing the early and late infection stages,respectively.1994 of the 3458 differentially expressed genes were upregulated at the early infection stage,whereas 1666 of the 3031 differentially expressed genes were upregulated at the late infection stage.The functional enrichment analysis indicated that the plant cell wall biogenesis was significantly induced at the early infection stage,while the callose deposition response was more active at the late infection stage.The callus deposition-mediated defense response was mainly composed of the cytochrome P450 family.Genes involved in the synthesis and regulation of phytoalexin coumarin were effectively activated.F6’H1 and S8 H,encoding key enzymes in the biosynthesis of coumarins and their derivatives,were more strongly expressed at the late infection stage than at the early infection stage.In addition,our study found that PME17 showed the high upregulation(log2foldchange =25.7)at the late infection stage,the gene encoding pectin methylesterase that regulates methyl esterification of pectin in plant cell walls,while no significant changes at the early infection stage.2.The response patterns of the citrus phyllosphere microbiome due to D.citri infectionThe collective community-level change of phyllosphere microbiome in response to D.citri infection was profiled by 16 S metabarcoding,shotgun metagenomics combined with co-occurrence network and genomic binning analysis.Multiple microbiome features suggested a shift in citrus phyllosphere microbiome upon D.citri infection,highlighted by the marked reduction of community evenness in the endophytic compartment(decrease from 0.50 to 0.36),the emergence of large numbers of new bacterial ASVs in the epiphytic compartment,and the intense microbial network(average degree =11.95).We also identified the microbiome features from functional perspectives in infected leaves,such as enriched microbial functions for metabolism(e.g.,carbohydrate and amino acid),iron competition and potential antifungal traits,and enriched Sphingomonas spp.that present beneficial genomic-characteristics.Sphingomonas spp.were found to be the main contributor for the functional enrichment of “iron complex outer membrane receptor protein” in the infected leaves.Among the“iron complex outer membrane receptor protein”,53.7% of non-redundant genes from the metagenomic dataset were annotated as Sphingomonas.3.The antagonistic activity of phyllosphere bacteria against D.citri infectionNinety-four bacterial ASVs were cultivated and identified by the two-sided barcode PCR system.Twelve bacteria associated with the phyllosphere microbiome shift were further investigated the microbial function.These bacteria were then tested in dual culture assays for their antagonism against D.citri.Seven of the 12 bacterial isolates exhibited different antagonistic activities against D.citri.Sphingomonas asv20,Pantoea asv90,and Methylobacterium asv41 showed strong antagonistic activities,and the percent inhibition of radial growth values were 40.8 ± 4.2%,39.2 ± 1.2%,and 36.8± 1.5%,respectively.The above bacterial isolates as well as Mucilaginibacter asv102 were further tested to determine their disease suppression activity against D.citri in the phyllosphere in glasshouse experiments.Overall,treatment with these isolates conferred protection against D.citri infection as compared with the non-treated control at different levels,with reductions in disease index ranging from 65.7 to 88.4%.Moreover,Sphingomonas asv20 showed a stronger suppression ability against D.citri in iron-deficient condition than in iron-sufficient condition.Genomic analysis supported the role of iron competition during Sphingomonas antagonistic action.Sphingomonas asv20 has 99 genes encoding Ton B-dependent receptors,45 of which are annotated as “iron complex outer membrane receptor protein”.Our study profiled the response patterns of citrus host and their phyllosphere microbiome in response to D.citri infection by multi-omics analysis,revealed the transcriptional evidence to support the defense mechanism of citrus host against D.citri infection,and identified the potential mechanisms for citrus phyllosphere microbiome cope with D.citri challenge.Overall,our findings provide novel insights into understanding the microbiome–pathogen–plant interactions.