Igwe, David O.

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.

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.