Citrus Huanglongbing Diagnosis Based on Molecular Detection of Associated Liberibacter Species

Citrus huanglongbing (HLB) is one of the most devastating citrus diseases in the world. The disease is associated with three species of Candidatus Liberibacter, „Ca. L. asiaticus‟, „Ca. L. africanus‟, and „Ca. L. americanus‟, and transmitted mainly by the Asian citrus psyllid (Diaphorina citri) and the African citrus psyllid (Trioza erytreae). Upon the first report of the disease in São Paulo, Brazil in 2004 and Florida, USA in 2005, HLB started to devastate the two largest citrus industries in the world. The disease continues to rapidly spread to the remaining citrus producing areas still free of the disease, such as Central America and Middle East, besides its presence in almost all Asian citrus producing nations. Early detection of the associated bacterium in host plants and vector insects is essential to HLB control and management. Sampling, sample processing and testing are three major processes in disease detection. The main objective of this invited talk is to compile recent advances in method development of sampling, sample processing and molecular testing of Candidatus Liberibacter species associated with the disease, and to make recommendations to increase the efficiency and ability to detect the associated bacterium in survey samples. Based on uneven distribution of the bacterium in plant tissues associated with irregular vector transmission, low bacterial titer during early stages of infection in host plants and vector insects, and recent quantitative data of the bacterial populations in infected plants and vectors, different sampling methods and sample sizes are recommended for host plant and vector insect surveys. These recommendations are useful for surveys in areas with HLB and HLB-like symptomatic, asymptomatic orchards and nurseries. In addition, adequate storage methods are also suggested for plant and insect samples prior to testing for the associated bacterium. Sample processing methods, especially of the bacterial DNA isolation from suspect plant and insect samples were compared. A closed mechanical sample homogenization system with commercial DNA extraction kits using DNA binding filters was highly advised for critical samples to avoid sample cross contamination and to obtain consistently high yield and purity of the bacterial DNA. Positive internal control primer and/or probe sets are proscribed for real-time monitoring on DNA extraction quality and PCR testing efficiency of Liberibacter species from suspect plant and insect samples. In comparison with traditional diagnostic methods including electronic microscopy, ELISA, use of fluorescent substance and starch accumulation in host plants, disease symptomology, and biological indexing, isotheral DNA amplification including the Loop-mediated isothermal amplification (LAMP) and the cycleave isothermal and chimeric primer-initiated amplification with probe technology (Cycleave ICAN), single and nested conventional PCR, and single and multiplex real-time PCR assays were analyzed for specificity, sensitivity and assay performance. Multiplex quantitative real-time PCR methods using the species-specific primers and/or probes for associated bacterium and the positive internal control primers and/or probes targeting the host plant DNA or vector psyllid DNA were appraised for HLB detection. The potential of 125 selected genes from the complete genome of „Ca. L. asiaticus‟ was also discussed for future use in multiple-loci detection.