Publications

4467

Psyllids are vectors of fastidious plant pathogenic ‘Candidatus Liberibacter’ species that infect both the psyllid vector and plant host. Understanding the molecular and cellular basis of ‘Ca. Liberibacter’ interactions with the psyllid host will aid in identification of effectors involved in invasion and multiplication and facilitate transmission to the host plant. The differential expression of previously identified genes/loci with predicted involvement in tomato host–plant– ‘Ca. L. solanacearum’–prophage–Wolbachia endosymbiont dynamics was quantified by RT-qPCR amplification. Fifteen ‘Ca. Liberibacter solanacearum genes and/or prophage loci and four predicted Wolbachia spp. loci were analyzed in potato psyllids in a 14-day time-course study, post-48-h acquisition-access period by potato psyllids on ‘Ca. L. solanacearum’-infected tomato plants. The ‘Ca. L. solanacearum’-infected tomato host plants were used as an infected host ‘calibrator’ species lacking involvement of psyllid effectors. ‘Ca. L. solanacearum’ genes with predicted functions in adhesion, motility, transport, and virulence that are associated with the prophage lysogenic lifestyle were differentially expressed. In contrast, the prophage-loci expression was synchronous with early or late phase of psyllid-‘Ca. L. solanacearum’ infection, respectively. The observations are consistent with the previously in silico-predicted ‘Ca. L. solanacearum’ gene and prophage/Wolbachia loci functions and time-course global expression patterns. Knockdown of ‘Ca. L. solanacearum’ genes involved in invasion, biofilm formation, and colonization would be expected to impair the vertical and horizontal transmission of ‘Ca. L. solanacearum’ to psyllid offspring and host plants, respectively.

A Gram-negative, facultative anaerobic, rod-shaped, motile with peritrichous flagella, fluorescent bacterium, designated ‘Candidatus Pseudomonas auctus’ sp. nov. JDE115, was isolated from soybean root nodules in Virginia and characterized using a comprehensive integrative methodology. Growth of JDE115 occurred with 0–5.0% (w/v) NaCl (optimum 1%), at pH 6.0–10.0 (optimum pH 7.0), and at 10–40°C (optimum 28°C) in LB broth. Phylogenetic analyses based on the 16S rRNA gene placed the isolate as a member of a novel species within the genus Pseudomonas. Phylogenetic analyses based on whole-genome sequences, 16S rRNA, showed JDE115 having the highest similarity to Pseudomonas glycinae MS586. Average Nucleotide Identity (ANI) analysis also revealed the highest similarity of JDE115 to Pseudomonas glycinae MS586 (94.59%), which is below the 95% threshold for species delineation. Genome-to-genome distance analysis (GGDC, Formula 2) showed a maximum value of 57.10% with the same strain, far below the 70% cutoff. The primary isoprenoid quinone detected in JDE115 was ubiquinone-9 (Q-9) and the DNA G + C content was 60.68 mol%. The whole-cell fatty acid profile was dominated by C16:0, C17:0 cyclo, and the summed features 3 (C16:1ω7c and/or C16:1ω6c) and 8 (C18:1ω7c and/or C18:1ω6c). Additional fatty acids detected included 12:0, 14:0, and 18:0. Based on these phenotypic, chemotaxonomic, and phylogenetic data, strain JDE115 is proposed to represent a new species in the genus Pseudomonas, for which the name ‘Candidatus Pseudomonas auctus’ sp. nov. is proposed.

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.