Heck, Michelle


Publications (12)

Strain tracking of ‘<i>Candidatus</i> Liberibacter asiaticus’, citrus greening disease pathogen, enabled by high-resolution microbiome analysis of the Asian citrus psyllid

Citation
Higgins et al. (2022). Phytopathology®
Names
Ca. Liberibacter asiaticus
Subjects
Agronomy and Crop Science Plant Science
Abstract
The Asian citrus psyllid, Diaphorina citri, is an invasive insect and a vector of 'Candidatus Liberibacter asiaticus' (CLas), a bacterium whose growth in Citrus species results in huanglongbing (HLB), also known as citrus greening disease. Methods to enrich and sequence CLas from D. citri often rely on biased genome amplification and nevertheless contain significant quantities of host DNA. To overcome these hurdles, we developed a simple pre-treatment DNase and filtration (hereafter PDF) protocol to remove host DNA and directly sequence CLas and the complete, primarily uncultivable, microbiome from D. citri adults. The PDF protocol yielded CLas abundances upwards of 60% and facilitated direct measurement of CLas and endosymbiont replication rates in psyllids. The PDF protocol confirmed our strains derived from a progenitor Florida CLas strain and accumulated 156 genetic variants, underscoring the utility of this data for bacterial strain tracking. CLas genetic polymorphisms arising in lab-reared psyllid populations included prophage encoding regions with key functions in CLas pathogenesis, putative antibiotic resistance loci, and a single secreted effector. These variants suggest laboratory propagation of CLas may result in different phenotypic trajectories among laboratories, and may confound CLas physiology or therapeutic design and evaluation if these differences remain undocumented. Finally, we obtained genetic signatures affiliated with Citrus nuclear and organellar genomes, entomopathogenic fungal mitochondria, and commensal bacteria from laboratory-reared and field-collected D. citri adults. Hence, the PDF protocol can directly inform agricultural management strategies related to bacterial strain tracking, insect microbiome surveillance, and antibiotic resistance screening.

Direct DNA sequencing of ‘<i>Candidatus</i> Liberibacter asiaticus’ from <i>Diaphorina citri</i>, the Asian citrus psyllid, and its implications for citrus greening disease management

Citation
Higgins et al. [posted content, 2022]
Names
Ca. Liberibacter asiaticus
Abstract
AbstractThe Asian citrus psyllid, Diaphorina citri is an invasive insect 1 and a vector of ’Candidatus Liberibacter asiaticus’ (CLas), a bacterium whose growth in Citrus species results in citrus greening disease 2,3. Methods to enrich and sequence CLas from D. citri often rely on biased genome amplification 4 and nevertheless contain significant quantities of host DNA 5,6. To overcome these hurdles, we developed a simple pre-treatment DNase and filtration (hereafter PDF) protocol to directly sequence CLas and the complete, primarily uncultivable, microbiome from D. citri adults. The PDF protocol yielded CLas abundances upwards of 60% and enabled detection of 156 genetic variants in these strains compared to progenitor strains in Florida, which included prophage encoding regions with key functions in CLas pathogenesis, putative antibiotic resistance loci, and a single secreted effector. These variants suggest laboratory propagation of CLas may result in different phenotypic trajectories among laboratories, and may confound CLas physiology or therapeutic design and evaluation if these differences remain undocumented. Finally, we obtained genetic signatures affiliated with Citrus nuclear and organellar genomes, entomopathogenic fungal mitochondria, and commensal bacteria from laboratory-reared and field-collected D. citri adults. Hence, the PDF protocol can inform agricultural management strategies related to pathogen evolution 7, insect microbiome surveillance 8, antibiotic resistance screening 9, and gut content analysis 10.

Host Plant Adaptation Drives Changes in Diaphorina citri Proteome Regulation, Proteoform Expression, and Transmission of ‘Candidatus Liberibacter asiaticus’, the Citrus Greening Pathogen

Citation
Ramsey et al. (2022). Phytopathology® 112 (1)
Names
Ca. Liberibacter asiaticus
Subjects
Agronomy and Crop Science Plant Science
Abstract
The Asian citrus psyllid (Diaphorina citri) is a pest of citrus and the primary insect vector of the bacterial pathogen, ‘Candidatus Liberibacter asiaticus’ (CLas), which is associated with citrus greening disease. The citrus relative Murraya paniculata (orange jasmine) is a host plant of D. citri but is more resistant to CLas compared with all tested Citrus genotypes. The effect of host switching of D. citri between Citrus medica (citron) and M. paniculata plants on the acquisition and transmission of CLas was investigated. The psyllid CLas titer and the proportion of CLas-infected psyllids decreased in the generations after transfer from CLas-infected citron to healthy M. paniculata plants. Furthermore, after several generations of feeding on M. paniculata, pathogen acquisition (20 to 40% reduction) and transmission rates (15 to 20% reduction) in psyllids transferred to CLas-infected citron were reduced compared with psyllids continually maintained on infected citron. Top-down (difference gel electrophoresis) and bottom-up (shotgun MS/MS) proteomics methods were used to identify changes in D. citri protein expression resulting from host plant switching between Citrus macrophylla and M. paniculata. Changes in expression of insect metabolism, immunity, and cytoskeleton proteins were associated with host plant switching. Both transient and sustained feeding on M. paniculata induced distinct patterns of protein expression in D. citri compared with psyllids reared on C. macrophylla. The results point to complex interactions that affect vector competence and may lead to strategies to control the spread of citrus greening disease.

An Excised Leaf Assay to Measure Acquisition of ‘<i>Candidatus</i> Liberibacter asiaticus’ by Psyllids Associated with Citrus Huanglongbing Disease

Citation
Igwe et al. (2022). Phytopathology® 112 (1)
Names
Ca. Liberibacter asiaticus
Subjects
Agronomy and Crop Science Plant Science
Abstract
Huanglongbing, or citrus greening disease, is the most serious disease of citrus worldwide and is associated with plant infection by ‘Candidatus Liberibacter asiaticus’ (CLas) and other Liberibacter species. CLas is transmitted by Diaphorina citri, the Asian citrus psyllid, in a circulative propagative manner. Circulative propagative transmission is a complex process comprising at least three steps: movement of the pathogen into vector tissues, translocation and replication of the pathogen within the vector host, and pathogen inoculation of a new host by the vector. In this work, we describe an excised leaf CLas acquisition assay, which enables precise measurements of CLas acquisition by D. citri in a streamlined laboratory assay. Briefly, healthy fourth and fifth instar D. citri nymphs acquire CLas from excised CLas-positive leaves, where the insects also complete their developmental cycle. CLas titer in the resulting adults is measured using quantitative PCR and CLas-specific 16S rRNA gene primers. We observed positive correlations between CLas titer in each leaf replicate and the CLas titer that developed in the insects after acquisition (rs = 0.78; P = 0.0002). This simple assay could be used to detect CLas acquisition phenotypes and their underlying genotypes, facilitate assessment of plant factors that impact acquisition, and screen for compounds that interfere with CLas acquisition by delivering these compounds through the excised leaf.

Multi-omics Comparison Reveals Landscape of Citrus limon and Citrus sinensis Response to ‘Candidatus Liberibacter asiaticus’

Citation
Chin et al. (2021). PhytoFrontiers™ 1 (2)
Names
Ca. Liberibacter asiaticus
Subjects
General Medicine
Abstract
Comparison of the metabolic changes prior to symptom development upon infection with Candidatus Liberibacter asiaticus (CLas), the bacterium associated with citrus greening disease, between citrus hosts with different tolerances is lacking. The objective of this study was to compare the early response of Lisbon lemon (Citrus limon) and Washington navel orange (Citrus sinensis [L.] Osbeck), two citrus species commercially important to California, to CLas through graft inoculation. Here, we compare the transcriptome, proteome, and metabolome response, using RNA sequencing, liquid chromatography tandem mass spectrometry, and 1H nuclear magnetic resonance, respectively, from our two recently published studies examining the response of the lemon and navel oranges separately, and introduce new micronutrient data from inductively coupled plasma mass spectrometry analysis, focusing on lemons at 10 and 14 weeks postgrafting (wpg), and navels at 8 and 18 wpg, prior to symptom development. Several micronutrients accumulated in presymptomatic infected lemons compared with controls, whereas little change was observed in the navels. Photosynthesis proteins were substantially altered by CLas infection in navels, with fewer changes observed in lemons. The metabolome differed between control and infected navels throughout infection, although differences between control and infected lemons were identified only after symptom expression. Taken together, these findings highlight differences in response to CLas between two varieties with differing tolerances. [Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .