Lou, Binghai

Abstract Citrus Huanglongbing (HLB) is one of the most devastating diseases in the citrus industry and is caused primarily by ‘Candidatus Liberibacter asiaticus’ (CLas), a phloem-restricted gram-negative bacterium transmitted via citrus psyllids in the field. Precise CLas detection is crucial for HLB control, particularly during extensive surveys in new emerging regions. Unfortunately, the lack of a practical on-site detection method for CLas due to the limited specificity of immunostrips and the costive non-user friendliness approaches prevent the effective disease control. In this study, a probe-based recombinase polymerase amplification (RPA) targeting the CLas five-copy nrdB gene (β-subunit of ribonucleotide reductase, RNR) was developed and evaluated for its specificity, sensitivity, and reliability. To enhance user-friendliness and facilitate widely use, an All-in-one kit was premade as freeze-dried pellets in individual tubes, containing all necessary components except DNA template. The applicability of the All-in-one kit for CLas detection was confirmed using plant or psyllid crude DNA extracts. Collectively, the All-in-one kit offers a specific, sensitive, rapid, cost-effective, and practical alternative for diagnosing CLas in field conditions when coupled with simplified crude DNA extraction method, providing an alternative approach for on-site CLas detection in the context of HLB.

Huanglongbing (HLB) is one of the most destructive diseases in citrus production worldwide. Early detection of HLB pathogens can facilitate timely removal of infected citrus trees in the field. However, low titer and uneven distribution of HLB pathogens in host plants make reliable detection challenging. Therefore, the development of effective detection methods with high sensitivity is imperative. This study reports the development of a novel method, tandem repeat-based polymerase chain displacement reaction (TR-PCDR), for the detection of ‘Candidatus Liberibacter asiaticus’, a widely distributed HLB-associated bacterium. A uniquely designed primer set (TR2-PCDR-F/TR2-PCDR-1R) and a thermostable Taq DNA polymerase mutant with strand displacement activity were used for TR-PCDR amplification. Performed in a regular thermal cycler, TR-PCDR could produce more than two amplicons after each amplification cycle. Sensitivity of the developed TR-PCDR was 10 copies of target DNA fragment. The sensitive level was proven to be 100× higher than conventional PCR and similar to real-time PCR. Data from the detection of ‘Ca. L. asiaticus’ with filed samples using the above three methods also showed similar results. No false-positive TR-PCDR amplification was observed from healthy citrus samples and water controls. These results thereby illustrated that the developed TR-PCDR method can be applied to the reliable, highly sensitive, and cost-effective detection of ‘Ca. L. asiaticus’.