Fu, S. M.

‘ Candidatus Liberibacter asiaticus’ ( CL as) is associated with the most destructive disease of citrus, huanglongbing ( HLB ). The most widely used methods for detection of CL as 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 CL as 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 CL as infection varied widely from one sample to another, but the patterns were highly correlated among serial sections from the same sample, suggesting that CL as preferentially colonizes adjacent phloem cells in a vertical rather than horizontal direction.

Huanglongbing (HLB), also known as citrus greening, is currently the most destructive citrus disease. Anatomical analyses of HLB-affected sweet orange were carried out by light and electron microscopy. As compared with healthy citrus, the phloem plasmodesmata were plugged with callose, and in some samples the phloem was collapsed. Chloroplast structures were deformed. Prophage sequences occupy a significant portion of the genome of ‘Candidatus Liberibacter asiaticus’ and have been used to distinguish strains from Yunnan and Guangdong provinces in China and Florida. Interestingly, a large number of possible putative phage particles were observed attached on the surface of ‘Ca. L. asiaticus’ cells in plants inoculated with strain FJ3 from Fujian Province, China. Phage particles have been observed previously only in periwinkle plants artificially inoculated in Florida with ‘Ca. L. asiaticus’ that carried the SC1-type prophage. PCR assays verified the presence of the SC1-type prophage sequences previously described from this bacterium in Florida in the FJ3 isolate. This is the first time that suspected phage particles have been observed in sweet orange trees infected with ‘Ca. L. asiaticus.’