A Study on High-Throughput Technologies for the Identification of Citrus Pathogens and the Characterization of Host Responses to Infections
- Author(s): Dang, Tyler
- Advisor(s): Vidalakis, Georgios
- et al.
High-throughput technologies for citrus diagnostic and characterization of host response in response to infection were evaluated. The high-throughput extraction using the MagMax 96 (Thermo Fisher Scientific, Waltham, MA) was found to be comparable to other existing RNA extractions methods for viroid and viruses testing. The method was adopted by the Citrus Clonal Protection Program and immediately utilized for nursery stock testing and field surveys. This resulted in significantly lowering the virus and viroid infections in the CA nursery stock program and the led to the discovery of citrus viroid V (CVd V) and citrus leaf blotch virus (CLBV), two exotic pathogens of California (CA). I was able to confirm the presence of CVd V by fulfilling Koch’s postulate through biological indexing and sanger sequencing. Alternatively, CLBV was confirmed by high-throughput sequencing (HTS). I expanded the HTS for the detection of citrus pathogens and utilized the e-probe diagnostic nucleic acid analysis (EDNA) online platform to simplify the data analysis. As proof-of-concept e-probes were developed for citrusviii tristeza virus (CTV), citrus exocortis viroid (CEVd), and Candidatus Liberibacter asiaticus (CLas). The e-probes worked as well as the currently approved diagnostic tools such as polymerase chain reaction (PCR). HTS was also applied for the characterization of citrus response in response to citrus dwarfing viroid (CDVd) infection. I discovered 4 conserved (3 from and 1 roots) and 3 novel miRNAs (stem only). The miRNA target host functions are associated with plant development and cellular growth that is consistent with the observed dwarfing phenotype in sweet orange trees on trifoliate rootstocks. In summary, the present dissertation provides in-depth application of high throughput methods for citrus diagnostics and characterization of host response to infection. These studies lead to the application of important high-throughput tools which have been utilized by the CCPP and provide insights into viroid-host interaction for future research.