An Excised Leaf Assay to Measure Acquisition of ' Liberibacter asiaticus' by Psyllids Associated with Citrus Huanglongbing Disease.

Huanglongbing, or citrus greening disease, is the most serious disease of citrus worldwide and is associated with plant infection by ' Liberibacter asiaticus' (Las) and other species. Las is transmitted by , the Asian citrus psyllid, in a circulative propagative manner. Circulative propagative transmission is a complex process comprising at least three steps: movement of the pathogen into vector tissues, translocation and replication of the pathogen within the vector host, and pathogen inoculation of a new host by the vector. In this work, we describe an excised leaf Las acquisition assay, which enables precise measurements of Las acquisition by . in a streamlined laboratory assay. Briefly, healthy fourth and fifth instar . nymphs acquire Las from excised Las-positive leaves, where the insects also complete their developmental cycle. Las titer in the resulting adults is measured using quantitative PCR and Las-specific 16S rRNA gene primers. We observed positive correlations between Las titer in each leaf replicate and the Las titer that developed in the insects after acquisition ( = 0.78; = 0.0002). This simple assay could be used to detect Las acquisition phenotypes and their underlying genotypes, facilitate assessment of plant factors that impact acquisition, and screen for compounds that interfere with Las acquisition by delivering these compounds through the excised leaf.