Targeted Early Detection of Citrus Huanglongbing Causal Agent ' Candidatus Liberibacter asiaticus' Before Symptom Expression.

Citrus Huanglongbing (HLB) is the most severe disease of citrus plants caused by ' Candidatus Liberibacter asiaticus' and transmitted by the insect vector Asian citrus psyllid (ACP). No effective curative measure is available against HLB. For citrus production areas without HLB or with low HLB disease incidence, removal of ' Ca. L. asiaticus' inoculum is critical to prevent HLB spread. Such a strategy requires robust early diagnosis of HLB for inoculum removal to prevent ACP acquisition and transmission of ' Ca. L. asiaticus'. However, early diagnosis of HLB is challenging, because the citrus trees remain asymptomatic for several months to years after ' Ca. L. asiaticus' transmission by ACP. In this study, we report a new method for targeted early detection of ' Ca. L. asiaticus' in cultivar Valencia sweet orange ( Citrus sinensis ) before HLB symptom expression. We take advantage of the fact that ' Ca. L. asiaticus' remains around the ACP feeding site immediately after transmission into the young flush and before flush maturation. ACPs secrete salivary sheaths at their feeding sites, which can be visualized using Coomassie brilliant blue staining owing to the presence of salivary sheaths secreted by ACP. Epifluorescence and confocal microscopy indicate the presence of salivary sheaths beneath the blue spots on ACP-fed leaves. Quantitative real-time polymerase chain reaction (PCR) and conventional PCR assays are able to detect ' Ca. L. asiaticus' in the ACP feeding surrounding areas as early as 2 to 20 days after ACP feeding. This finding lays a foundation to develop much-needed tools for early diagnosis of HLB before symptom expression, thus assisting ' Ca. L. asiaticus' inoculum removal and preventing HLB from spreading.