Detection of ‘Candidatus Liberibacter asiaticus’ in citrus by concurrent tissue print based qPCR and Immunoassay

Authors: FU, SHIMIN - Southwest University; LIU, HUAWEI - Southwest University; LIU, Q - Southwest University; ZHOU, CHONGYANG - Southwest University; Hartung, John

Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/10/2018
Publication Date: 2/5/2019
Citation: Fu, S., Liu, H., Liu, Q.H., Zhou, C., Hartung, J.S. 2019. Detection of ‘Candidatus Liberibacter asiaticus’ in citrus by concurrent tissue print based qPCR and Immunoassay. Plant Disease. https://doi.org/10.1111/ppa.12998.
DOI: https://doi.org/10.1111/ppa.12998

Interpretive Summary: ‘Candidatus Liberibacter asiaticus’ (CLas), a bacterium, is associated with the most destructive disease of citrus, huanglongbing (HLB). The most widely used methods to detect the pathogen require the isolation of CLas DNA from the plant sample to be tested. Isolation of CLas DNA is laborious and inefficient because the pathogen is located inside woody plant cells that are difficult to break apart. Once the plant cells are broken open the DNA must be purified using chemical methods. Overall, testing for CLas is laborious, inefficient, expensive and generates chemical waste. We have developed and combined two different methods to detect CLas without first purifying the pathogen’s DNA. In our methods, the pathogen is deposited on an inexpensive paper by pressing a cut stem or leaf onto the surface of the paper. Then CLas can be detected with an antibody based method called immune tissue printing. Alternatively, the DNA can be washed off the surface of the paper with a simple buffer and tested for the presence of CLas. Both methods are inexpensive and scale to large numbers of samples. Because the methods combine two independent methods, antibodies and DNA based detection, they complement each other very well. Regulatory officials, researchers, and testing facilities will find our methods useful for the detection of CLas and management of citrus greening disease.

Technical Abstract: ‘Candidatus Liberibacter asiaticus’ (CLas) is associated with the most destructive disease of citrus, huanglongbing (HLB). The most widely used methods for detection of CLas are PCR-based and require purification of DNA from plant samples. Elution of DNA from tissue prints made on nitrocellulose membranes followed by qPCR (TPE-qPCR) was compared to DNA extraction of plant tissue followed by qPCR (X-PCR) by testing the same tissue samples. The former estimated higher CLas population in tissue prints than the latter (t-test; p = 0.009). All extracts prepared for TPE-qPCR throughout the experiment were also tested by conventional PCR and 80.8% were identified positive. A similar set of stem and petiole tissue samples were tested by TPE-qPCR and immunoassay. Although the detection rate by TPE-qPCR was higher than by immunoassay, about 6% of tissue prints were positive by immunoassay but not by TPE-qPCR. Thus, a higher detection rate will be achieved by combining TPE-qPCR with immunoassay. Significant differences were observed in the performance of nitrocellulose membranes from different manufacturers in these assays. Immune tissue prints showed that the spatial distribution pattern of CLas infection varied widely from one sample to another, but the patterns were highly correlated among serial sections from the same sample, suggesting that CLas preferentially colonizes adjacent phloem cells in a vertical rather than horizontal direction.