Heck, Michelle

Huanglongbing (HLB, or citrus greening disease) affects all citrus varieties world-wide. In the USA, Asia, and South America the causal agent is “Candidatus Liberibacter asiaticus” (CLas), a phloem-limited, uncultured, alphaproteobacterium. The hemipteran insect vector, Diaphorina citri (Asian citrus psyllid) acquires and transmits CLas in a circulative, propagative manner. In addition to CLas, D. citri hosts multiple symbiotic bacterial species including Wolbachia (wDi). In D. citri, wDi has been sequenced and studied but specific roles in D. citri biology are unknown. Using well-established quantitative PCR methods we measured CLas titer in D. citri collected from four groves in central Florida with distinct HLB management strategies and tested whether CLas and wDi titer were correlated in a sub-set of these insects. Grove site had the largest effect on CLas titer. Sex had no effect on CLas titer, while higher wDi titer was correlated with non-infected insects. Our results suggest that more directed follow-up research is necessary and important to clarify whether field management tactics influence CLas titer in D. citri and to better understand gene-by-environment interactions among D. citri, wDi and CLas. Now that millions of trees in Florida have been treated with injectable formulations of oxytetracycline, which may decrease bacterial populations in D. citri, this study may represent the last biologically meaningful snapshot of grove-level vector-pathogen ecology in the state during the HLB epidemic.

AbstractHuanglongbing (HLB, or citrus greening disease) affects all citrus varieties world-wide. In the USA, Asia, and South America the causal agent is “CandidatusLiberibacter asiaticus” (CLas), a phloem-limited, uncultured, alphaproteobacterium. The hemipteran insect vector,Diaphorina citri(Asian citrus psyllid) acquires and transmitsCLas in a circulative, propagative manner. In addition toCLas,D. citrihosts multiple symbiotic bacteria includingWolbachia(wDi). InD. citri, wDi has been sequenced and studied but specific roles inD. citribiology are unknown. Using well established quantitative PCR methods we measuredCLas titer inD. citricollected from four groves in central Florida with distinct HLB management strategies and tested whetherCLas and wDi titer were correlated in a sub-set of these insects. Grove site had the largest effect onCLas titer. Sex had no effect onCLas titer, while higher wDi titer was correlated withCLas-uninfected insects. Our results suggest that more directed follow-up research is necessary and important to clarify whether field management tactics influenceCLas titer inD. citriand to better understand gene-by-environment interactions amongD. citri, wDi andCLas. Now that millions of trees in Florida have been treated with injectable formulations of oxytetracycline, which is likely to decrease bacterial populations inD. citri, this study may represent the last biologically meaningful snapshot of grove-level vector-pathogen ecology in the state during the HLB epidemic.

Huanglongbing (HLB), referred to as citrus greening disease, is a bacterial disease impacting citrus production worldwide and is fatal to young trees and mature trees of certain varieties. In some areas, the disease is devastating the citrus industry. A successful solution to HLB will be measured in economics: citrus growers need treatments that improve tree health, fruit production, and most importantly, economic yield. The profitability of citrus groves is the ultimate metric that truly matters when searching for solutions to HLB. Scientific approaches used in the laboratory, greenhouse, or field trials are critical to the discovery of those solutions and to estimate the likelihood of success of a treatment aimed at commercialization. Researchers and the citrus industry use a number of proxy evaluations of potential HLB solutions; understanding the strengths and limitations of each assay, as well as how best to compare different assays, is critical for decision-making to advance therapies into field trials and commercialization. This perspective aims to help the reader compare and understand the limitations of different proxy evaluation systems based on the treatment and evaluation under consideration. The researcher must determine the suitability of one or more of these metrics to identify treatments and predict the usefulness of these treatments in having an eventual impact on citrus production and HLB mitigation. As therapies advance to field trials in the next few years, a reevaluation of these metrics will be useful to guide future research efforts on strategies to mitigate HLB and vascular bacterial pathogens in other perennial crops.

AbstractThe Asian citrus psyllid,Diaphorina citri, is a vector of ‘CandidatusLiberibacter asiaticus’ (CLas), a gram-negative, obligate biotroph whose infection inCitrusspecies is associated with citrus greening disease, or Huanglongbing (HLB). Strategies to blockCLas transmission byD. citriremain the best way to prevent the spread of the disease into new citrus growing regions. However, identifying control strategies to block HLB transmission poses significant challenges, such as the discovery and delivery of antimicrobial compounds targeting the bacterium and overcoming consumer hesitancy towards accepting the treatment. Here, we computationally identified and tested a series of 20-mer nodule-specific cysteine-rich peptides (NCRs) derived from the Mediterranean legume,Medicago truncatulaGaertn. (barrelclover) to identify those peptides that could effectively prevent or reduceCLas infection in citrus leaves and/or preventCLas acquisition by the bacterium’s insect vector,D. citri. A set of NCR peptides were tested in a screening pipeline involving three distinct assays: a bacterial culture assay, aCLas-infected excised citrus leaf assay, and aCLas-infected nymph acquisition assay that includedD. citrinymphs, the only stage ofD. citri’s life-cycle that can acquireCLas leading to the development of vector competent adult insects. We demonstrate that a subset ofM. truncatula-derived NCRs inhibit bothCLas growth in citrus leaves andCLas acquisition byD. citrifromCLas-infected leaves. These findings reveal NCR peptides as a new class and source of biopesticide molecules to controlCLas for the prevention and/or treatment of HLB.

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 pretreatment DNase and filtration (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 upward of 60% and facilitated direct measurement of CLas and endosymbiont replication rates in psyllids. The PDF protocol confirmed our lab strains derived from a progenitor Florida CLas strain and accumulated 156 genetic variants, underscoring the utility of this method 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 that laboratory propagation of CLas could result in different phenotypic trajectories among laboratories and could 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.

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.

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.

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.

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 .