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.

Figure 1

DNA sequence alignment of the mitochondrial 16S locus from Diaphorina citri (ACP) and other psyllids used for primer design for the ACP 16S LAMP assay. Grey shading highlights ACP. Primers are indicated above the alignment.

Figure 2

Maximum Likelihood tree (5,000 bootstrap replicates) developed to confirm species identifications and assess the genetic variation in the sequence of mitochondrial cytochrome oxidase, subunit I (COI) between ACP and other Australian native and exotic psyllids sequenced in this study. Bootstrap values < 50% are not reported. The target species, D. citri, is indicated in bold at the top of the tree, distinct from the other taxa. The scale bar is a genetic distance of 5%. All COI sequences were generated in this study, GenBank accession numbers are indicated together with the VAITC database identification number.

Figure 3

Amplification profile and anneal derivative curve comparison of ACP gBlock gene fragment (synthetic positive control), and samples of D. citri. (a) Amplification profile of D. citri samples at 16 and 22 min and gBlock 1 × 10⁶ copies/µL, at ~ 8 min (pink). Negative, no amplification (purple). (b) Anneal derivative of LAMP amplicons showing two peaks, ~ 78.5 °C for D. citri and ~ 83 °C for the gBlock (pink).

Figure 4

Optimised ACP 16S LAMP assay results using 1:8:4 primer ratio. (a) Positive amplification profile of 5 D. citri samples within 10 to 20 min and one non-target Trioza species no amplification (blue). Negative flat line (purple). (b) Anneal derivative at ~ 78.5 °C for 5 D. citri samples. Negative and Trioza species, no anneal peak (purple and blue).

Figure 5

Amplification profile and anneal derivative of Candidatus Liberibacter asiaticus (CLas) LAMP assay. (a) Amplification profile of CLas DNA (synthetic positive) within 15 min (pink). (b) Anneal derivative at ~ 85.5 °C for CLas positive (pink). All the 6 D. citri samples tested were negative, no amplification (flat line). Negative, no amplification and no anneal peak (purple).