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This is a general protocol for the vegetative propagation of citrus material infected with the bacterium Candidatus Liberibacter asiaticus (CLas), the presumed causal agent of citrus greening disease (HLB), using an aeroponic cloning apparatus. The following procedure details a workflow using an EZ-Clone Pro Low Cloning System from Hydrobuilder.com. This system comes in varying cell-sizes (number of clones). We have found the most versatile to be the 16- or 32-cell size based on ease of preparation, keeping algae growth to a minimum, and maintenance. Cloning versus seed sowing has been useful in producing infected material for experimental assays requiring cloned, mature citrus trees as opposed to genetically variable plants from seeds. Where seed germination can take up to 12+ months to achieve plants with stem diameters ≥ 10 mm and 5+ years for flowering, infected branches from flowering trees can be directly propagated using this protocol within a few months and retain their maturity. Clone viability rate differs by citrus cultivar and general health of the cutting used, with citron and CLas negative material having the highest success rate. Infected citron cuttings have had a 95% survival rate, regardless of the diameter of the cutting. The cutting diameter ranged from 2mm-12mm and was limited to 12mm maximum, which is the size of the collar on the EZ-Clone Pro Low cloner.

Citrus Huanglongbing (HLB), caused by “Candidatus Liberibacter asiaticus” (CLas), is a destructive disease threatening global citrus industry. Although citrus cultivars differ in HLB sensitivity, how infection alters endophytic bacterial communities in cultivars with contrasting susceptibility remains unclear. Here, we compared endophytic microbiome shifts in leaf and root tissue of HLB-susceptible Shatangju mandarin (C. reticulata cv. Shatangju) and HLB-tolerant Shatian pomelo (C. maxima cv. Shatian) at 10 months post grafting (MPG) with CLas-infected buds. Infected Shatangju mandarin showed severe symptoms and higher CLas levels in leaf than root, while infected Shatian pomelo remained asymptomatic with higher CLas in root than leaf. High-throughput 16S rRNA gene sequencing revealed that CLas infection restructured endophytic microbial community in tissue-specific manner. CLas-infected Shatian pomelo leaf displayed increased microbial diversity and network complexity, while CLas-infected Shatangju mandarin root harbored more disease-suppressive taxa like Streptomyces. Across both cultivars, network complexity correlated inversely with CLas abundance. Key genera (e.g., Pseudomonas, Streptomyces, Prevotella) correlated positively with CLas, suggesting roles in mitigating pathogen effects. Predicted functional profiling indicated activated pathways (amino acid metabolism, terpenoid/polyketide biosynthesis, xenobiotic biodegradation) potentially supporting host defense in infected tissues. The HLB tolerance of Shatian pomelo appeared linked to tissue-specific bacterial restructuring, particularly recruitment of antibiotic-producing bacteria and enhanced metabolic resilience. These findings elucidated cultivar-specific microbial strategies against CLas, offering insights for microbiome-mediated HLB management.

This study reports the draft genome of ‘ Candidatus Phytoplasma pyri’ strain P1, isolated from Argentina. The genome assembly consisted of 17 contigs, with a total length of 575,431 bp, a GC content of 20.35%, and 125× coverage. A total of 537 genes were annotated, including those related to metabolism, genetic information processing, and signaling. Phylogenetic analysis placed ‘ Ca. Phytoplasma pyri’ within the 16SrX group, supporting its classification as a distinct species from ‘ Ca. Phytoplasma mali’ and ‘ Ca. Phytoplasma prunorum’, with average nucleotide identity values ranging from 90.3 to 90.5%. In contrast, strain P1 shares 98.1% average nucleotide identity with the Chilean strain ‘ Ca. Phytoplasma pyri’ UCh_1, supporting their classification as members of the same species. The investigation also identified 12 secreted proteins, including homologs of known effectors, as well as conserved proteins associated with virulence mechanisms. A homolog of the immunodominant membrane protein was also characterized, revealing conserved gene organization across related strains. These findings contribute to the understanding of ‘ Ca. Phytoplasma pyri’ pathogenicity and provide valuable genomic data for future research. [Formula: see text] Copyright © 2025 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .