New excised-leaf assay method to test inoculativity of Asian citrus psyllid (Hemiptera: Psyllidae) with Candidatus Liberibacter asiaticus associated with citrus huanglongbing disease.

The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), is the primary vector of Candidatus Liberibacter asiaticus (Las) associated with huanglongbing, or citrus greening, the most devastating citrus (Citrus spp.) disease worldwide. Here, we developed a new "excised-leaf assay" that can speed up Las-inoculativity tests on Asian citrus psyllid from the current 3-12 mo (when using whole citrus seedlings for inoculation) to only 2-3 wk. Young adults of Asian citrus psyllid that had been reared on Las-infected plants were caged on excised healthy sweet orange [Citrus sinensis (L.) Osbeck] leaves for a 1-2-wk inoculation access periods (IAP), and then both psyllids and leaves were tested later by quantitative polymerase chain reaction (PCR). When single adults were tested per leaf, percentages of Las-positive leaves averaged 2-6% by using HLBaspr primers and 10-20% by using the more sensitive LJ900 primers. Higher proportions of Las-positive leaves were obtained with 1) higher densities of inoculating psyllids (5-10 adults per leaf), 2) longer IAPs, and 3) incubation of leaves for 1 wk postinoculation before PCR. Logistic regression analysis indicated a positive correlation between Las titer in Asian citrus psyllid adults tested singly and the probability of detecting Las in the inoculated leaves, correlations that can be very useful in epidemiological studies. Comparison between excised leaves and whole seedlings, inoculated consecutively for 1 wk 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, materials, and greenhouse space, and it may enhance vector relation and epidemiological studies on Las and potentially other Liberibacter spp. associated with huanglongbing disease.