Chemotherapeutic Control And Metagenomic Analysis Of Citrus Huanglongbing

Citrus Huanglongbing （HLB） or greening is the most devastating disease of citrus. HLB has been spread to the most of the citrus growing areas worldwide and causing significantly losses or decline in both the production and the profit of citrus industry. Control of HLB in the field is urgently needed but difficult due to a lack of resistant citrus species and cultivars as well as an effective integrated disease management. Chemotherapy is considered to be an effective short-term strategy for controlling HLB. However, Since Ca. L. asiaticus （Las） resides in citrus phloem and only a small portion of effective compounds may reach the target sites, chemotherapy usually fail to kill the pathogen. It has become critically important to develop a delivery system for the effective compound to migrate efficiently and work against Las effectively inside HLB-affected citrus plants. In the meantime, it is also important to monitor the dynamics of Las bacterial microbiome in response to the chemical compounds and heat treatment through Phylochip-based metagenomic analyses.In chapter 2, a transcuticular nanoemulsion formulation was developed to enhance the permeation of an effective antimicrobial compound （ampicillin; Amp） against HLB through the citrus cuticle into the phloem via a foliar spray. The results demonstrated that efficiency of cuticle isolation using an enzymatic method （pectinase and cellulase） was dependent on the citrus cultivar and Las-infection, and it was more difficult to isolate cuticles from valencia orange（Citrus sinensis） and HLB-symptomatic leaves. Of eight adjuvants tested, Brij 35 provided the greatest increase in permeability of the HLB-affected cuticle with a 3.33-fold enhancement of cuticular permeability over water control. An in vitro assay using Bacillus subtilis showed that nanoemulsion formulations containing Amp （droplets size=5.26±0.04 nm and 94±1.48 nm） coupled with Brij 35 resulted in greater inhibitory zone diameters （5.75 mm and 6.66 mm） compared to those of Brij 35 （4.34 mm） and Amp solution （2.83 mm） alone. Furthermore, the nanoemulsion formulations eliminated Las bacteria in HLB-affected citrus in planta more efficiently than controls. Our study shows that a water in oil （W/O） nanoemulsion formulation may provide a useful model for the effective delivery of chemical compounds into citrus phloem via a foliar spray for controlling citrus HLB.In chapter 3, based on various physiochemical characteristics of oils, surfactants, and organic solvents, a novel oil-in-water （O/W） nanoemulsion was optimized to combat citrus HLB. The nanoemulsion was produced using a spontaneous emulsification method for the efficient delivery of ampicillin into citrus phloem by applying it to bark. The nanoemulsion prepared from cremophor（?） EL （viscous oil）, acetone （water miscibility organic solvent）, and Span 80/Tween 80 （surfactant） exhibited a small droplet size （13.68±0.26 nm）. It also had an improved absorption rate, including a 2 days peak concentration （tmax）,71.9 ng/g maximum concentration （Cmax）, and 274.63% relative bioavailability （RBA） of ampicillin in HLB-affected citrus compared to the Amp solution alone （tmax= 6 days, Cmax= 56.4 ng/g and RBA=100%）. The same nanoemulsion was used to deliver five antimicrobials to control citrus HLB through the bark. We found that the nanoemulsion enhanced the therapeutic efficiency of validoxylamien A （increased by 57.58%）, combination of actidone and validoxylamine A （increased by 18.06%）, and sulfadimoethoxine sodium （increased by 22.22%） against Las. Therefore, this study provides an efficient self-nanoemulsifying delivery system for controlling citrus HLB.In chapter 4, two applications （trunk absorption and soil drench） and thermotherapy at three different temperatures were evaluated for the efficacy of five antimicrobial compounds. The results indicated that thermotherapy of HLB diseased citrus at 45℃ was most effective in reducing Las populations in plant phloem tissue. The high temperature also enhanced the delivery efficiency of effective chemical compounds into citrus phloem. In addition, Ampicillin （Amp） and the mixture of actidione and validoxylamine A （Act+VA） were most effective in inhibiting Las bacterium. When Amp or Act+VA were used alone with thermotherapy at 45℃, the area under disease progress curve standardized （AUDPCs） was significantly greater than that of thermotherapy treatment alone. In the trial of evaluating the application methods for chemo-thermotherapy of HLB diseased citrus, we also found that the bark absorption method was much better than the soil drench application. The Las population was greatly reduced, while AUDPCs and therapeutic efficiency （TE） of Amp and Act+VA treatments applied by bark absorption, was much greater in the bark absorption experiment. Therefore, we strongly believe that a chemo-thermotherapy through a bark absorption method can be used to combat citrus HLB. The research has also provided sufficient and useful information on how to conduct a successful chemo-thermotherapy in citrus HLB control.In chapter 5, Bacterial microbiome inside HLB-infected citrus that were treated with thermotherapy （45℃ and 40℃） and chemotherapy with sulfathiazole sodium （STZ） or sulfadimethoxine sodium （SDX） was characterized by Phylochip-based metagenomics. The Phylochip-based DNA sequence analyses determined that 331 empirical Operational Taxonomic Units （eOTUs） were detected in 26 phyla. Among them, Cyanobacteria spp （18.01%） were dominant in the bacterial population after thermo-chemotherapy treatment. The thermotherapy at 45℃ reduced eOTUs to 40.61% in leaves, much better in comparison with the treatment at 40℃ （to 48.08% eOTUs） and without thermotherapy （to 56.97% eOTUs）. The 45℃ thermotherapy also reduced the biodiversity of different bacterial families, especially the eOTU in phylum Proteobacteria that was reduced significantly. The eOTU₂8 of "Candidatus Liberibacter" that was not detected in thermotherapy at 45℃. Both SDX and STZ enhanced the specific bacterial eOTUs that belongs to Family Streptomycetaceae, Desulfobacteraceae, Chitinophagaceae, and Xanthomonadaceae, which were antagonistic to plant pathogens. In addition, our results also indicated that the combination of thermotherapy at 45℃ and chemotherapy with STZ and SDX were more effective in HLB control disease than that of using thermotherapy or chemotherapy alone, or that of the combinations of chemotherapy thermotherapy at 40℃. In generally, our study has provided valuable information on developing more effective and eco-friendly strategies in combating HLB.Overall, our results have developed and evaluated several effective strategies for HLB control and provided insight information on the bacterial microbiome dynamics in HLB-infected citrus with the treatment of thermotherapy and chemotherapy.