Laboratory Protocols for Candidatus Liberibacter asiaticus Transmission Biology and Management Research.

Abstract

Candidatus Liberibacter asiaticus (CLas), the causal agent of Huanglongbing, is transmitted by the Asian citrus psyllid (Diaphorina citri) in a persistent, propagative manner. Reproducible transmission studies require not only controlled inoculation assays but also standardized propagation of citrus host plants, maintenance of D. citri colonies, and reliable pathogen detection. This study presents a comprehensive set of coordinated protocols that collectively support controlled CLas transmission experimentation. The workflow includes propagation of Citrus macrophylla for colony and assay use; generation and maintenance of CLas-infected citrus and D. citri colonies; synchronization of D. citri populations to enable CLas acquisition; and a controlled single-D. citri transmission assay with defined inoculation access periods, and a Dellaporta-based nucleic acid extraction method compatible with citrus and psyllid tissues. Quantitative PCR detection is integrated throughout to verify infection status, determine CLas titer, and monitor CLas movement and systemic colonization in citrus over time. By combining standardized plant production, vector infection systems, transmission assays, and molecular diagnostics, these coordinated methods provide a unified framework for testing hypotheses about vector competence, pathogen-vector interactions, microbial co-infections, host susceptibility, and environmental effects on transmission. Together, the procedures enable consistent and comparable assessment of CLas acquisition, dissemination, and transmission outcomes under controlled laboratory conditions. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Controlled laboratory method for assessing Candidatus Liberibacter asiaticus transmission by Diaphorina citri Support Protocol 1: Generation of Candidatus Liberibacter asiaticus-infected Citrus macrophylla and Diaphorina citri Basic Protocol 2: Dellaporta total nucleic acid extraction Basic Protocol 3: Citrus macrophylla propagation.