Global Metabolomics Analysis of Pakistani Citrus Cultivars Infected with Huanglongbing or Citrus Greening

Abstract

Citrus is a significant leading fruit crop in Pakistan, with the highest export volume of 370 million tons. HLB is a bacterial disease that has been a potential threat to global citrus production with no effective cure to date. To fill the gap of existing knowledge of CLas and its pathophysiological modification. The infection of CLas in citrus trees depends on the metabolites involved inside its metabolic pathways. This study used to determine the global metabolites involved in the disease citrus greening. The CLas infection in citrus cultivars was detected through qRT-PCR. The Ct value ranged from 36.3 or no Ct value for the healthy samples. Here we report the first metabolic profiling of the vast range of targetted metabolites of ten citrus cultivars of Pakistan via LC-MS analysis. Results have been verified via the Tukey test (Pr|t|), One-way ANOVA (p≤ 0.05), and MetaboAnalyst 5.0 tools (Volcano plot, PCA, 2D and 3D PLS-DA plots, heat maps, VIP scores plot, Permutation test). A total of 500 statistically verified metabolites were detected in the leaves. Negative ion mode indicated 57% of metabolites, whereas 43% were identified in positive ion mode with good separation. Potential differences among the global metabolites of varying classes included sugars, amino acids, organic acids, phenolic acids, organic acids, carboxylic and nucleic acids, and flavonoids in positive and negative ion modes. There was an approximately 50% chance in negative ions that known metabolites separating healthy and HLB-infected leaf samples. Many untargeted compounds were also detected which were not found in the LC-MS (MZmine freeware) database, indicating the possibility of identifying novel metabolites that could be used as molecular markers for HLB diagnosis and management. This study provides a broad picture of the accumulation of metabolites involved in citrus plants with citrus greening. The metabolomic profile indicated the possible changes at the maturing stage of the disease.