An excised leaf assay to measure acquisition of “candidatus liberibacter asiaticus” by psyllids associated with citrus Huanglongbing disease

Authors: IGWE, DAVID - Cornell University; Higgins, Steven; Heck, Michelle

Submitted to: Phytopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/7/2021
Publication Date: 5/14/2021
Citation: Igwe, D.O., Higgins, S.A., Heck, M.L. 2021. An excised leaf assay to measure acquisition of “candidatus liberibacter asiaticus” by psyllids associated with citrus Huanglongbing disease. Phytopathology. in press. https://doi.org/10.1094/PHYTO-03-21-0124-SC.
DOI: https://doi.org/10.1094/PHYTO-03-21-0124-SC

Interpretive Summary: Huanglongbing is a citrus disease involving citrus host trees, an insect vector Diaphorina citri, known as the Asian citrus psyllid, and a bacterial pathogen ‘Candidatus Liberibacter asiaticus.’ Huanglongbing, also known as citrus greening disease, is considered to be the most devastating of all citrus diseases, and there is currently no adequate control strategy. Tree-to-tree spread by the psyllid involves three major steps: acquisition from an infected tree, translocation and replication in the psyllid body, and inoculation into a new host plant. The development of management strategies to block transmission depends on scientists' ability to study and measure the effects of new therapies each of these transmission steps in a precise manner. In this work, ARS scientists developed an assay, called an excised leaf acquisition assay, to measure the acquisition step of the transmission process. The new assay reduces the time it takes to study interactions between the psyllid and an infected tree from several months to three weeks and make measurements on a precise step in the transmission process. This method will enable scientists to develop new ways of blocking the spread of citrus greening by preventing psyllid transmission of the citrus greening pathogen.

Technical Abstract: Huanglongbing (HLB), or citrus greening disease, is the most serious disease of citrus worldwide and is associated with plant infection by “Candidatus Liberibacter asiaticus” (CLas) and other Liberibacter species. CLas is transmitted by Diaphorina citri, 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 CLas acquisition assay, which enables precise measurements of CLas acquisition by D. citri in a streamlined lab assay. Briefly, healthy 4th and 5th instar D. citri nymphs acquire CLas from excised CLas-positive leaves, where the insects also complete their developmental cycle. CLas titer in the resulting adults is measured using qPCR and CLas-specific 16S rDNA gene primers. We observed positive correlations between CLas titer in each leaf replicate and the CLas titer that developed in the insects after acquisition (rs = 0.78, P = 0.0002). This simple assay could be used to detect CLas acquisition phenotypes and their underlying genotypes, facilitate assessment of plant factors that impact acquisition, and screen for compounds that interfere with CLas acquisition by delivering these compounds through the excised leaf.