Zheng, Desen

Citrus huanglongbing (HLB) is recognized as the most destructive bacterial disease affecting citrus. Among the causal agents, Candidatus Liberibacter asiaticus (Las) is the predominant species causing HLB epidemics worldwide. Detecting this phloem limited bacterium at early or low titer stages and assessing its cellular activity remain major challenges for HLB research and management. To address these limitations, we developed a one step reverse transcription quantitative PCR (RT qPCR) assay for highly sensitive Las detection using total nucleic acids and the established Li 16S rRNA/rDNA primer–probe set. This method simultaneously amplifies both Las 16S rRNA and rDNA in a single reaction, increasing sensitivity by up to 500 fold compared with qPCR, which targets only 16S rDNA. The improved sensitivity reduced false negatives, expanded sampling capacity, and enabled detection of low titer infections previously undetectable. In addition, the higher abundance of 16S rRNA generated a consistent Ct gap between RT qPCR and qPCR, which we used to estimate relative Las cell activity in vivo in host insects and plants and to identify the most effective inoculum sources. This comparative detection strategy was further applied to evaluate inoculum potential, monitor changes in relative cell activity during in vitro cultures, and assess antimicrobial treatments targeting Las. The persistent detection of long lasting, low titer Las infections underscores the complex biology of the HLB causal agent and highlights ongoing challenges for effective disease management.

AbstractCandidatusLiberibacter asiaticus (Las) is one of the causal agents of citrus huanglongbing (HLB) epidemics worldwide. Due to its fastidious nature, intracellular and systemic infection, detecting Las at early and/or low-titer infection, as well as differentiating between live or dead cells in the host psyllids and citrus plants is critical for effective HLB management. To achieve both sensitive Las detection and differentiation, we employed one-step reverse transcription-quantitative PCR (RT-qPCR) using total nucleic acids as template. This method allows use of both Las 16S rRNA and rDNA as template in the same reaction and increases detection sensitivity by up to 1000-fold in comparison with quantitative PCR (qPCR). The increased sensitivity significantly reduces false negative detection and detects the otherwise undetectable low-titer Las infections. Furthermore, the greater the abundance of 16S rRNA present in the samples, the bigger the Ct gap obtained between RT-qPCR and qPCR results. The numerical Ct gap can be used to deduce relative Las cellular activity and indirectly infer whether cells are alive or dead. In addition, this comparative detection method also can be used to select inoculum and monitor relative cell activity duringin vitroLas culture and evaluate the effectiveness of antimicrobial treatments against Las bacteria.

Abstract Candidatus Liberibacter asiaticus (Las) is one of the causal agents of huanglongbing (HLB), the most devastating disease of citrus worldwide. Due to the intracellular lifestyle and significant genome reduction, culturing Las in vitro has proven to be extremely challenging. In this study, we optimized growth conditions and developed a semi-selective medium based on the results of nutritional and antibiotic screening assays. Using these optimized conditions, we were able to grow Las in the LG liquid medium with ca.100- to 1000-fold increase, which peaked after 4 to 6 weeks and were estimated to contain 106 to 107 cells/ml. The cultured Las bacteria remained in a dynamic state of growth for over 20 months and displayed limited growth in subcultures. The survival and growth of Las was confirmed by fluorescence in situ hybridization with Las-specific probes and expression of its metabolic genes. Growth of Las in the optimized medium relied on the presence of a helper bacterium, Stenotrophomonas maltophilia FLMAT-1 that is multi-drug resistant and dominant in the Las co-culture system. To recapitulate the disease, the co-cultured Las was inoculated back to citrus seedlings via psyllid feeding. Although the Las-positive rate of the fed psyllids and inoculated plants were relatively low, this is the first demonstration of partial fulfillment of Koch’s postulates with significant growth of Las in vitro and a successful inoculation of cultured Las back to psyllids and citrus plants that resulted in HLB symptoms. These results provide new insights into Las growth in vitro and a system for improvement towards axenic culture and anti-Las compound screening.