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Title: Global Metabolomics Analysis of Pakistani Citrus Cultivars Infected with Huanglongbing or Citrus Greening
Authors: Khadija Gilani, Asma Ahmed, Rehana Badar, Shaista Javaid, Mehmood ul Hassan, Shagufta Naz, Sami Ullah
Journal: International Journal of Phytopathology
Publisher: Center for Community Learning
Country: Pakistan
Year: 2023
Volume: 12
Issue: 2
Language: English
DOI: 10.33687/phytopath.012.02.4620
Keywords: CitrusHuanglongbingHLBLC-MSCandidatus liberibactermetabolomic profilingcitrus greening
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 C Las and its pathophysiological modification. The infection of C Las 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 C Las 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.
To determine the global metabolites involved in Citrus Greening Disease (HLB) caused by Candidatus Liberibacter asiaticus (CLas) infection in ten Pakistani citrus cultivars using LC-MS profiling.
Leaves from ten CLas-infected and ten healthy citrus cultivars were collected. CLas infection was confirmed using qRT-PCR (Ct values ranged from 36.3 or no Ct value for healthy samples). Anatomical modifications were observed using Scanning Electron Microscopy (SEM), focusing on starch content and phloem plugging. Global metabolomic profiling was performed on lyophilized leaf samples using Liquid Chromatography-Mass Spectrometry (LC-MS) in both negative and positive ion modes. Statistical analysis included Tukey test, One-way ANOVA (p ≤ 0.05), Volcano plots, PCA, 2D and 3D PLS-DA plots, heat maps, VIP scores plot, and Permutation tests using MetaboAnalyst 5.0 and SAS software.
graph TD; A[Collection of Healthy & CLas-Infected Leaf Samples] --> B[CLas Infection Confirmation]; B -- qRT-PCR --> C[Starch Content Analysis via SEM]; C --> D[LC-MS Metabolomic Profiling +/- Ion Modes]; D --> E[Statistical Analysis: ANOVA, PLS-DA, Volcano Plot]; E --> F[Identification of Metabolite Differences]; F --> G[Conclusion on Metabolic Markers & Defense Pathways];
The study confirms that HLB infection significantly alters the metabolome of citrus leaves, particularly affecting carbohydrate metabolism (starch accumulation and sugar depletion) and amino acid profiles, which are linked to plant defense mechanisms (e.g., phenylalanine precursor for phenylpropanoid pathway). The observed metabolic shifts, including changes in flavonoids and phenolic compounds, suggest altered defense responses and potentially contribute to fruit quality changes (e.g., bitter taste). The metabolic profiling provides potential molecular markers for early and late-stage HLB diagnosis and management.
A total of 500 statistically verified metabolites were detected in the leaves (57% in negative ion mode, 43% in positive ion mode). Significant differences were found across metabolite classes including sugars, amino acids, organic acids, phenolic acids, carboxylic acids, nucleic acids, and flavonoids. HLB-infected leaves showed statistically significant higher total starch content (p=0.001) compared to healthy leaves. Several amino acids (e.g., L-phenylalanine, L-methionine, L-tyrosine) and phenolic compounds (e.g., hesperidin) were upregulated in infected samples, while sugars/carbohydrates (e.g., glucose, fructose) and some nucleic acids decreased. SEM confirmed phloem plugging and starch accumulation in infected tissues.
HLB infection profoundly impacts the metabolomic profile of citrus leaves, leading to starch accumulation and disruption of carbohydrate transport via phloem impairment. Understanding these metabolic pathways, particularly the regulation of defensive metabolites like amino acids and phenolics, offers a basis for developing disease-tolerant citrus varieties and novel control strategies.
1. The Ct value range for healthy samples was reported as 36.3 or no Ct value.
2. A total of 500 statistically verified metabolites were detected in the leaves.
3. Negative ion mode identified 57% of the detected metabolites, while positive ion mode identified 43%.
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