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

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 PCR (X‐PCR) by testing the same tissue samples. The former estimated a 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 as positive. A similar set of stem and petiole tissue samples was 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 would be achieved by combining TPE‐qPCR with immunoassay. Significant differences were observed in the performance of nitrocellulose membranes from different manufacturers in these assays. Immunotissue 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.