LAMP assay for the detection of the Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psylloidea: Psyllidae)

Abstract

Diaphorina citri Kuwayama, also known as the Asian citrus psyllid (ACP), can vector the bacterium Candidatus Liberibacter asiaticus (CLas), agent of Huanglongbing (HLB): an incurable disease affecting citrus trees worldwide. In citrus growing regions where ACP and HLB are absent, such as Australia, the risk of an incursion and consequent economic damage to citrus industries make this psyllid one of the top-priority pests. Due to ACP’s small dimensions and the generally poorly studied native psylloid fauna worldwide, morphological identification of this insect to distinguish it from harmless species is challenging, especially in the field, and with immature, partial or damaged specimens. To allow rapid and efficient detection of ACP in the field, we designed and optimised a new Loop-mediated isothermal amplification (LAMP) assay for the detection of D. citri based on the mitochondrial 16S locus. The optimised ACP 16S LAMP assay produced amplification from D. citri samples within 13.3 ± 3.6 min, with an anneal derivative of ~ 78.5 °C. A synthetic gBlock gene fragment was also developed to be used as positive control for the new LAMP assay with a different anneal derivative of ~ 83 °C. An existing commercially available LAMP assay for detection of the bacterium CLas was also tested in this study on ACP DNA. The ACP 16S LAMP assay we developed and tested here provides a valuable new in-field compatible tool that can allow early detections of ACP, enabling a quick biosecurity response, and could potentially be adopted by a wide range of users, from farmers to agronomists and from researchers to industry.