Submitted to: Journal of Economic Entomology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 11/9/2012
Publication Date: 2/12/2013
Publication URL: http://handle.nal.usda.gov/10113/57056
Citation: Ammar, E., Walter, A., Hall, D. 2013. A new excised-leaf assay method to test the inoculativity of the Asian citrus psyllid with Candidatus Liberibacter asiaticus associated with citrus huanglongbing disease. Journal of Economic Entomology. 106:25-35. Interpretive Summary: The Asian citrus psyllid (ACP) is the primary vector of the bacterium associated with huanglongbing, or citrus greening, the most devastating citrus disease worldwide. However, normally, only a low percentage of ACP can actually inoculate this bacterium into healthy plants after acquiring it from diseased citrus. Thus, to understand the epidemiology of this disease, it is important to assess the inoculativity of psyllids in any given population. Here, we developed a new ‘excised-leaf assay’ that can speed up inoculativity tests on ACP from the current 3-12 months (when using whole citrus seedlings for inoculation) to only 2-3 weeks. This new excised-leaf assay method saves considerable time, material and greenhouse space, and may hopefully enhance vector-relation and epidemiological studies on this and probably other bacterial species associated with huanglongbing disease.
Technical Abstract: The Asian citrus psyllid (ACP, Diaphorina citri, Hemiptera: Psyllidae) is the primary vector of Candidatus Liberibacter asiaticus (Las) associated with huanglongbing, or citrus greening, the most devastating citrus disease worldwide. Here, we developed a new excised-leaf assay that can speed up Las-inoculativity tests on ACP from the current 3-12 months (when using whole citrus seedlings for inoculation) to only 2-3 weeks. Young adults of ACP that had been reared on Las-infected plants were caged on excised healthy sweet orange leaves for 1-2 wk inoculation access periods (IAP), and both psyllids and leaves were tested later by quantitative PCR. When single adults were tested per leaf, percentages of Las-positive leaves averaged 2-6 percent using HLBaspr primers, and 10-20 percent using the more sensitive LJ900 primers. Higher proportions of Las-positive leaves were obtained with the following: 1) higher densities of inoculating psyllids (5-10 adults/leaf), 2) longer IAP, and 3) incubation of leaves for 1-wk post-inoculation before PCR. Logistic regression analysis indicated a positive correlation between Las titer in ACP adults tested singly and the probability of detecting Las in the inoculated leaves, which can be very useful in epidemiological studies. Comparison between excised leaves and whole seedlings, inoculated consecutively for 1 week each by one or a group of psyllids, indicated no significant difference between Las detection in excised leaves or whole plants. This new ‘excised-leaf assay’ method saves considerable time, material and greenhouse space, and may hopefully enhance vector-relation and epidemiological studies on Las and probably other Liberibacter spp. associated with huanglongbing disease.