Aster Yellows Phytoplasma, Aster Leafhopper And Canola: Development And Application Of Improved Molecular Methods For Pathogen Detection And Genetic Characterization Provide Increased Understanding Of Aster Yellows Disease

Phytoplasmas are insect-vectored, difficult-to-culture bacterial pathogens that infect a wide variety of plants. They are associated with diseases connected with severe yield losses in global agricultural production, including Aster Yellows (AY). Aster Yellows phytoplasma (AYp) is primarily transmitted by the aster leafhopper (ALH), Macrosteles quadrilineatus Forbes, and AY outbreaks in Western Canada tend to damage canola crops most severely. Even though there are several methods to control AYp spread, the most common method is insecticide spray, which can damage the environment if used unsustainably. Tools that can rapidly diagnose phytoplasma infection and accurately identify phytoplasma strains are of critical importance for disease management. Currently, detecting AYp involves a time-consuming process of transporting insect samples and extracting DNA, and this method often delays the application of mitigative measures. A rapid and field-adaptable diagnostic method was developed, which uses Flinders Technology Associates (FTA) PlantSaver paper cards to extract insect DNA followed by a loop-mediated isothermal amplification (LAMP) assay. This approach successfully detected AYp in under an hour, and its application could be expanded to a wide range of insect-transmitted pathogens. Disease management can also be improved by identifying and understanding the various species, strains, groups, and subgroups of phytoplasma. PCR-based methods targeting universal taxonomic markers (e.g., 16S rRNA) are commonly used to identify phytoplasmas in plant and insect tissues; however, these methods provide limited resolution of phytoplasma strains. In response to these limitations, a PCR-independent, hybridization-based multilocus sequence typing (MLST) assay was developed to precisely characterize phytoplasmas through the concurrent sequencing of seven taxonomic markers. This novel approach could serve as a standardized method for phytoplasma identification and may inform the understanding of phytoplasma spread in crop plants worldwide. Little is conclusively known about the long-distance dispersal patterns of ALH, but gaining a more comprehensive understanding of the species could positively influence the development of AY control strategies. A panel of 22 microsatellite markers for ALH was developed and used in multiplex format to explore the genetic makeup of Saskatchewan ALH populations. This initial investigation into ALH genetics indicated a wide range of genetic variation within populations. In addition, there was no correlation between genetic and geographic distances, suggesting that Western Canada is a melting pot for North American ALH populations. While this study is a pioneering work and cannot be compared to reference data, it is a critical step in furthering knowledge of ALH. In the context of Western Canada, while researchers explore knowledge gaps in AYp identification and ALH origins, this work has determined that the best approach to AY management is, and may remain, prompt detection of AYp using field-adaptable molecular diagnostic methods.

Name thesis:degree_name

Doctor of Philosophy (Ph.D.)

Level thesis:degree_level

Doctoral

Discipline thesis:degree_discipline

Biology

Grantor

University of Saskatchewan

Year dc:date.issued

2024

Author dc:creator

• Pusz-Bochenska, Karolina H

Advisors dc:contributor.advisor

• Wist, Tyler
• Gray, Jack

Committee members dc:contributor.committeemember

• Philiips, Iain
• Prager, Sean
• Yangdou, Wei
• Niyogi, Som
• Groves, Russell
• Dumonceaux, Tim

• aster yellows disease
• phytoplasma
• aster leafhopper
• molecular diagnostics

Language dc:language.iso

en

Handle dc:identifier.uri

https://hdl.handle.net/10388/15778

OAI identifier oai:identifier

oai:harvest.usask.ca:10388/15778