Molecular Approaches for Biological Control of X-Disease Leafhopper Vectors in Washington State

Cesar Alejandro Reyes Corral

X-disease phytoplasma remains one of the most significant threats to stone fruit production in the Pacific Northwest, with Colladonus leafhoppers serving as the primary vectors. However, vector ecology, symbiotic interactions, and the potential role of natural enemies remain poorly understood, limiting the development of sustainable management strategies. This dissertation integrates field surveys, molecular diagnostics, and biological control investigations to improve our understanding of Colladonus leafhoppers and their associated microbial communities. Seasonal surveys of Colladonus populations across multiple Washington sites revealed clear temporal and spatial dynamics, providing critical information to refine phenology models and improve vector monitoring. Molecular and morphological analyses led to the first documented record of Tomosvaryella big-headed flies in Washington State, confirming their parasitism of Colladonus species. In addition, a previously unrecognized yeast-like Hirsutella symbiont was discovered in C. reductus and C. geminatus. Evidence of vertical transmission and genetic divergence between populations suggests that this symbiont may influence vector physiology and potentially affect X-disease transmission dynamics. To enable comprehensive detection of vectors, pathogens, and natural enemies, a five-target qPCR multiplex assay was developed. This molecular tool allows simultaneous detection of C. geminatus, C. reductus, X-disease phytoplasma, Tomosvaryella lepidipes, and Dryinidae pincer wasps, facilitating high-throughput screening of field-collected specimens. The application of this assay has revealed valuable insights into vector-parasitoid interactions and natural enemy prevalence. Collectively, this research advances our understanding of Colladonus ecology, highlights the potential role of microbial symbionts in vector biology, and identifies previously unrecognized biological control agents. The development of molecular tools, including species-specific primers and the multiplex assay, provides a framework for integrating molecular diagnostics with field-based surveys. These findings lay the groundwork for future research aimed at exploiting biological control and microbial symbionts to mitigate X-disease outbreaks and improve sustainable vector management strategies.

Molecular Approaches for Biological Control of X-Disease Leafhopper Vectors in Washington State

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