Unraveling the metabolite signature of citrus showing defense response towards Candidatus Liberibacter asiaticus after application of endophyte Bacillus subtilis L1-21

Huanglongbing (HLB) is one of the most serious citrus diseases, caused by phloem limited endophytic bacteria Candidatus Liberibacter asiaticus (Clas), affecting worldwide citrus production. Metabolomics approaches were employed to gain insight into mechanisms involved in defense against Clas in endophyte Bacillus subtilis L1-21 treated diseased and healthy citrus plants. Using LC-ESI-MS/MS, we compared the metabolic profile of citrus plants before and after treatment with endophyte L1-21. Our analysis indicated large differences in citrus metabolites after endophyte L1-21 application. In total, seven hundred and fourty two metabolites were detected with highest percentage recorded for organic acids, flavone, amino acid derivatives, flavone C-glycosides, nucleotide derivatives, and flavonol. Interestingly, differentially expressed metabolites (DEMs) analysis revealed the amino acids, such as lysine and tyrosine which are involved in plant defense agianst pathogen attack were regulated in diseased citrus plants after endophyte application (padj<0.05). In addition, other important metabolites up-regulated were xanthine, leucic acid, and α-Linolenic acid implicated in different plant defense pathways against Clas. Furhter, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed important pathways related to purine metabolism, biotin metabolism, and betalain biosynthesis, terpenoid-quinone biosynthesis, phenylalanine, tyrosine and lysine biosynthesis, isoflavonoid biosynthesis (padj<0.05). Taken together, this is the first study using native endophytes in diseased and healthy state of citrus which has proven to be useful in disease management by strengthening the defense of citrus to Clas pathogen.