Detection of Liberibacter asiaticus in a single infected Asian citrus psyllid adult or nymph: Impact of dilution with clean Asian citrus psyllids (Diaphorina citri) during extraction

Now that the presence of Huanglongbing (HLB) has been confirmed in California, protecting the state’s citrus industry through early detection of disease is essential in curtailing its spread. Because ‘Candidatus Liberibacter asiaticus’ (Las), the putative causal agent of HLB, accumulates in its vector, the Asian citrus psyllid (ACP), Diaphorina citri, Kuwayama, accurate testing of the insect is vital. Due to the fact that insect secondary metabolites interfere with downstream applications (1, 3) there is concern about the number of insects pooled in DNA extractions without compromising Las detection. The current USDA CPHST approved method of testing (2) limits the pooling size to five individual insects per extraction followed by the highly sensitive quantitative polymerase chain reaction (QPCR) detection technique. The QPCR reaction targets a region of the Las 16S ribosomal gene (4) and simultaneously the D. citri specific wingless (WGLS) gene (5) as an internal control for extraction efficiency. In contrast, the CDFA laboratory routinely pools up to 25 individual insects for DNA extraction, followed by the same QPCR detection. If pooling 25 individuals is indeed a safe practice all the labs currently limiting sample pooling to five individuals could save a substantial amount of time and money. We decided to approximate the pooling limit using single insect equivalents of DNA extracted from Las infected ACP from colonies at USDA-ARS in Fort Pierce, Florida pooled with intact ACP from clean UC Riverside, California, quarantine colonies. Depending on results we would follow these experiments with experiments using intact Las positive ACP