Abstract
Candidatus
Liberibacter asiaticus (
C
Las), 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
C
Las transmission experimentation. The workflow includes propagation of
Citrus macrophylla
for colony and assay use; generation and maintenance of
C
Las‐infected citrus and
D. citri
colonies; synchronization of
D. citri
populations to enable
C
Las 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
C
Las titer, and monitor
C
Las 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
C
Las 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