Metabolic response of sweet orange (Citrus sinensis) to Candidatus Liberibacter asiaticus inoculation via its vector, Diaphorina citri
- Lombardi, Rachel Lynn
- Advisor(s): Slupsky, Carolyn M
Huanglongbing (HLB), also known as citrus greening disease or yellow dragon disease, is the most destructive disease of citrus worldwide. In the United States, Huanglongbing is associated with infection of the phloem-restricted bacterium Candidatus Liberibacter asiaticus (CLas). This pathogenic agent is transmitted by the Asian citrus psyllid (ACP; Diaphorina citri Kuwayama) which feeds directly on the phloem of diseased citrus plants, ingesting CLas in the process, and transmits the pathogen into the phloem of a new tree during subsequent feeding. Symptoms of infection with CLas can take months to years to develop which allows asymptomatic trees to act as sources of inoculum for dissemination of the pathogen. When symptoms do appear, the disease is associated with asymmetric leaf chlorosis (‘yellow mottle’), twig dieback, sparsely foliated trees, and production of fruit that is lopsided, bitter, and green even when ripe; symptoms ultimately culminate with premature tree death.
To date, most efforts to understand the plant’s early response to CLas introduce the pathogen by grafting diseased plant material onto healthy trees instead of using the pathogen’s natural ACP vector. To fully understand HLB, it is necessary to know how citrus responds to the psyllid vector and how that response differs when ACP are harboring CLas and introducing it into the plant. The purpose of this dissertation was to address this under-explored area of literature by characterizing the early leaf response of greenhouse citrus trees to exposure to ACP with or without CLas (Chapter 2) and to assess the translatability of ACP-inoculation greenhouse studies to trends observed in HLB field studies (Chapter 3).
In the first study (Chapter 2), we used transcriptomics, proteomics, and metabolomics to identify how exposure to ACP with or without CLas impacts the metabolism of citrus leaves for one year. Two hundred CLas-positive or CLas-negative ACP were introduced to pairs of Madam Vinous sweet orange (Citrus sinensis (L.) Osbeck) trees. Two weeks later, collection of leaf samples began and took place every two weeks for 52 weeks. The resulting analysis utilized transcriptomic data from four timepoints, proteomic data from six timepoints, and metabolomic data from 14 timepoints to gain insights into the response of trees over time. The results of this study indicated that the initial plant response of ACP-vectored CLas infection is primarily due to exposure to ACP and to a lesser extent, the presence or absence of CLas. Additionally, it demonstrated the complexity of this disease by identifying only 17 transcripts, one protein, and no metabolites that were consistently differentially accumulated between trees exposed to CLas-positive or CLas-negative ACP during the first 16 weeks post-exposure to psyllids. By using a controlled greenhouse environment to investigate the citrus response, we were able to clearly characterize changes in citrus leaf cellular metabolism due to these stimuli.
To understand the translatability of our results to real-world situations, a subsequent investigation was carried out to compare the metabolomic results of our greenhouse study to CLas-infected citrus grove trees from Texas (Chapter 3). Metabolomic data from symptomatic grove trees and non-infected screenhouse trees was previously acquired from southern Texas and reported as part of a multi-laboratory investigation to evaluate different early HLB detection techniques. By comparing the metabolic trends in response to ACP-inoculated CLas infection in greenhouse versus field trees, it was possible to understand how well controlled greenhouse studies reflect disease progression relative to what is actually occurring in the field. Overall, the results of this dissertation identified changes in citrus leaf metabolism due to ACP with or without CLas (Chapter 2) and examined the overlap and discrepancies between ACP-transmitted CLas infection between greenhouse and field studies (Chapter 3). The outcomes of the investigations conducted here can be used to refine molecular targets for early disease detection, development of resistant cultivars, and to inform a broader understanding of plant-microbe and plant-vector interactions.