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Citrus huanglongbing (HLB) is one of the most devastating citrus diseases worldwide. It is associated with a phloem-restricted bacterium ‘<i>Candidatus</i> Liberibacter asiaticus’ and primarily transmitted by Asian citrus psyllid in Florida. Due to the uncultured bacterial pathogen, HLB early diagnosis relies on DNA-based methods like polymerase chain reactions (PCR) including real-time quantitative PCR (qPCR). Although estimating live bacterial population (LBP) is critical for HLB research, PCR has limitations on differentiating live and dead cells, thus tends to overestimate LBP in hosts. Propidium monoazide (PMA), a novel DNA-binding dye, has already been successfully used on many bacterial plant pathogens to effectively remove DNA from dead cells, but no applications on uncultured bacteria were reported. In this study, PMA-qPCR protocols were first optimized to work with plant and psyllid materials, respectively. Then, they were used to monitor LBP dynamics inside HLB positive citrus and non-citrus hosts monthly through an 18-month period. Different LBP developing patterns were observed, which could indicate different living micro-environments inside different hosts for HLB bacteria. This rapid qPCR method provides an accurate way to estimate LBP in HLB hosts, which in turn should benefit various researches like disease epidemiology and serves as a crucial component in HLB management. <p><p>Keywords: Bacteria-Phytoplasma-Spiroplasma-Fastidious Prokaryote, Fruits-Nuts, Citrus