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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.

In the fall of 2015 and 2016, during annual disease surveys conducted by the Pennsylvania Department of Agriculture, seven peach trees (Prunus persica) from a commercial orchard in Berks County, PA, were observed exhibiting symptoms consistent with phytoplasma infection. Symptoms included premature leaf yellowing and reddening, foliar epinasty, shotholes and vein reddening. Seven out of 700 peach trees in the orchard displayed these symptoms and were ultimately removed. Leaf and stem samples were collected from seven symptomatic trees as well as from five asymptomatic ones for further analysis. Total DNA was extracted using the Qiagen DNeasy Plant Kit. All DNA samples were initially screened using a phytoplasma-specific real-time PCR assay (Hodgetts et al., 2009). Phytoplasma was detected in all symptomatic plants, but in none of the five asymptomatic plants. Positive samples were subsequently used in conventional nested PCR targeting the 16S rRNA gene, using primer pairs P1/16S-SR followed by P1A/16S-SR (Deng & Hiruki, 1991; Lee et al., 2004). PCR products were cloned, and sequenced. Four representative sequences were deposited in GenBank under accession numbers PV770936 to PV770939. Sequence analysis using BLASTn (NCBI) and iPhyClassifier (Zhao et al., 2009) revealed that the phytoplasma strains, designated as PYP-PA03, 04, 06, and 07, shared between 97.5% (1,482/1,520 bp) and 97.63% (1,483/1519 bp) sequence identity with the ‘Candidatus Phytoplasma fraxini’ reference strain 16SrVII-A (AF092209), suggesting that the peach associated phytoplasma is a ‘Ca. P. fraxini’-related strain. However, virtual RFLP analysis of the 16S rDNA F2nR2 fragments revealed patterns distinct from all previously established 16Sr groups/subgroups. The closest similarity was observed with members of the 16Sr group VI, but similarity coefficients were below the accepted subgroup threshold (0.97), supporting the distinctiveness of these strains. To further characterize the PYP-PA phytoplasma, partial sequences of the tuf and secA genes were PCR-amplified, cloned, and sequenced following protocols by Makarova et al. (2012) and Dickinson & Hodgetts (2013). The resulting sequences were identical for each gene, and one sequence was deposited in GenBank under accession numbers PV769898 (tuf gene) and PV769899 (secA gene). BLASTn analysis of the tuf gene revealed 93.25% (470/504 bp) and 90.67% (457/504 bp) sequence identity with the homologous gene in the Argentinian alfalfa witches’-broom phytoplasma isolate ArAWB-2021 16SrVII-C (CP131022) and the ‘Ca. P. fraxini’ isolate AshY1 16SrVII-A (CP146843), respectively. Similarly, the secA gene shared 89.16% (691/775 bp) and 88.30% (679/769 bp) sequence identity with the same isolates ArAWB-2021 (CP131022) and AshY1 (CP146843), respectively. These collective results suggest that the PYP-PA phytoplasma is phylogenetically related to members of the Ash yellows group (16SrVII). In 2010, Zunnoon‐Khan et al. identified a phytoplasma strain PRU0430 of group 16SrVII associated with peach trees in Ontario, Canada (GU223903). To our knowledge, the present study is the first to report a ‘Ca. P. fraxini’-related strain associated with peach trees in the United States. These findings highlight the need for further research to fully characterize this potentially novel phytoplasma, which constitutes a distinct lineage from the one identified in Canada and underscore the importance of continued surveillance and molecular investigation of emerging phytoplasma-associated diseases in the region.

ABSTRACT The phyllody severity in sesame crop ranged from 9% to 20% under field conditions, and the maximum was recorded in Thiruvannamalai district of Tamil Nadu, India. The observed morphological characters of the transmitting insect vector Orosius albicinctus were light ochraceous colour with irregular striations and dark brown with black mottling. The infected sesame samples were subjected to PCR amplification by using universal primer pair P1/P6, followed by nested PCR using R16F2n/R16R2 primers, which amplified ~1.25 kb, which was also occurred in the Parthenium weed plant. Further, BLAST analysis showed that the sequences were aligned with the 16SrII‐B group phytoplasma consisting of Candidatus Phytoplasma aurantifolia (94.87%), sesame phyllody (97.97%), Blainvillea acmella phytoplasma (97.70%) and Mollugo disticha phyllody phytoplasma (98.28%). Since there is no adorus impact despite the vast host range, polyphagous insect vectors, lack of environmentally acceptable effective insecticide, and unavailability of resistant sources, management of phyllody disease has been attempted with biotic inducers (salicylic acid [SA], methyl jasmonate [MeJA] and beta amino butyric acid [BABA]) individually (50, 100 and 150 ppm) and in combination. Among the inducers tested in vitro, SA 50 ppm showed a higher vigour index (1401) in the seed infiltration method of seed treatment, further confirmed in blotter paper technique. The same treatment showed a high germination percentage (86%) with more shoot length on the 60th day (67.0 cm) and five branches/plant under glasshouse experiment. In addition, SA 50/100 ppm increased the phenolic activity and plant defence enzymes; hence it has been further test verified at two different field locations, and the results revealed that seed infiltration treatment with SA 50/100 ppm, followed by foliar application of SA (50/100 ppm) at 30, 45 and 60 DAS, was shown to be effective by recording lesser disease severity compared to control (42.2%). Also, salicylic acid treatment showed higher germination (87.6%), more capsules/plant, thereby incorporation of inducers like salicylic acid in the integrated disease management will serve as a viable and sustainable management option.

‘ Candidatus Phytoplasma pyri’, the pathogen associated with pear decline, affects pear trees across both the old and new worlds. However, research on this phytoplasma has been limited by the lack of genomic data. This study presents the first draft genome of ‘ Ca. P. pyri’ using a strain from Chile, with its genomic features analyzed in comparison with the closely related ‘ Ca. Phytoplasma’ species ‘ Ca. P. mali’ and ‘ Ca. P. prunorum’. The draft genome spans 456,478 bp with a GC content of 20.4%. Key genes possibly associated with pathogenicity and potential pathogenic effectors were identified, and they are notably lacking orthologs of known effectors. A single potential mobile unit similar to that of ‘ Ca. P. mali’ was identified. It is characterized by the absence of the transposase tra5 and the presence of the IS3 family transposase iSErh1. Multilocus sequence analysis of six genetic markers (16S rRNA gene, LSU36p, tuf, aceF, secA, and secY) from 10 Chilean and 10 Italian samples revealed high genetic uniformity among the Chilean strains, collected from five geographically distant orchards over a span of 13 months; by contrast, strains with greater diversity were detected among those from Italy, collected from a few localities over approximately 30 years. These findings suggest limited evolutionary divergence of this phytoplasma in Chile. This study provides a foundational framework for investigations into the pathogenic mechanisms and evolutionary dynamics of ‘ Ca. P. pyri’.

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 .