Metagenomic detection of novel bacterial combinations associated with citrus decline in Iraq

Citrus decline diseases pose significant threats to global fruit production, with complex bacterial pathogen interactions remaining poorly understood. In Iraq's Karbala governorate, severe citrus decline has affected orange orchards for 25 years, causing tree mortality within 3-5 years and substantial economic losses. PCR screening was performed on 75 symptomatic orange trees to detect phloem-limited bacterial pathogens, followed by whole-genome metagenomics on three selected PCR-positive samples to characterize associated microbial communities. Raw NGS reads from these three samples were quality-filtered, then MetaPhlAn2 was used to map reads to a curated marker database and identify bacterial, archaeal, viral, and eukaryotic taxa. The analysis revealed complex mixed infections involving three major plant bacterial pathogens: whereas PCR assays identified Candidatus Phytoplasma citri in 13.3% of the 75 samples, two additional phloem-limited pathogens, Ca. Liberibacter asiaticus and Spiroplasma sp., were exclusively detected via metagenomic sequencing across the three analyzed samples. Trimmed reads were assembled into contigs and analyzed phylogenomically against a global reference dataset. Genome assemblies yielded three for Ca. P. citri (576,881 bp, 424,689 bp, and 72,017 bp) and one each for Ca. L. asiaticus (1,151,288 bp) and Spiroplasma sp. (1,833,004 bp). These findings should be considered exploratory given the limited metagenomic sample size (n = 3); independent validation using targeted molecular approaches is required to confirm the presence of Ca. L. asiaticus and Spiroplasma sp. This is the first report documenting the metagenomic detection and characterization of a mixed infection involving Ca. Phytoplasma citri, Ca. Liberibacter asiaticus, and Spiroplasma sp. associated with citrus decline in Iraq. These findings provide crucial insights into pathogen populations and characterization and inform targeted management strategies for emerging bacterial diseases in Iraqi agricultural systems.