Rapid Detection of Pityophthorus juglandis (Blackman) (Coleoptera, Curculionidae) with the Loop-Mediated Isothermal Amplification (LAMP) Method

Abstract

The walnut twig beetle Pityophthorus juglandis is a phloem-boring bark beetle responsible, in association with the ascomycete Geosmithia morbida, for the Thousand Cankers Disease (TCD) of walnut trees. The recent finding of TCD in Europe prompted the development of effective diagnostic protocols for the early detection of members of this insect/fungus complex. Here we report the development of a highly efficient, low-cost, and rapid method for detecting the beetle, or even just its biological traces, from environmental samples: the loop-mediated isothermal amplification (LAMP) assay. The method, designed on the 28S ribosomal RNA gene, showed high specificity and sensitivity, with no cross reactivity to other bark beetles and wood-boring insects. The test was successful even with very small amounts of the target insect's nucleic acid, with limit values of 0.64 pg/µL and 3.2 pg/µL for WTB adults and frass, respectively. A comparison of the method (both in real time and visual) with conventional PCR did not display significant differences in terms of LoD. This LAMP protocol will enable quick, low-cost, and early detection of P. juglandis in areas with new infestations and for phytosanitary inspections at vulnerable sites (e.g., seaports, airports, loading stations, storage facilities, and wood processing companies).

Keywords: bark beetle; invasive species; molecular identification; thousand canker disease; walnut.

Figure 1

Real-time LAMP amplification curves from adults (green with circles) and frass (red with triangles) of WTB.

Figure 2

Capillary electrophoresis using the QIAxcel Capillary Electrophoresis System (QIAgen, Valencia, CA, USA) of end point PCR carried out with the primers 14F/125R on serial 1:5 dilutions of WTB adult DNA.

Figure 3

LoD assay of visual LAMP based on WTB adults using serial 1:5 dilutions (from 10 ng/µL to 5.12 fg/µL).

Figure 4

Capillary electrophoresis using the QIAxcel Capillary Electrophoresis System (QIAgen, Valencia, CA, USA) of end point PCR carried out with the primers 14F/125R on serial 1:5 dilutions of artificial DNA from WTB frass.

Figure 5

LoD assay of Visual LAMP based on artificial DNA from WTB frass using serial 1:5 dilutions (from 10 ng/µL to 5.12 fg/µL).

Figure 6

Black walnut branches infested by P. juglandis with an indication (red arrows) of the frass sampling sites.

Figure 7

Annealing sites of the LAMP primers on the 28S ribosomal RNA gene sequence of P. juglandis (accession number, KP201676.1): F3/B3 (black), LoopB/LoopF (purple), B2/F2 (red), F1c/B1c (blue).

Figure 8

Partial sequence alignment of the 28S ribosomal RNA gene resulting from the in-silico LAMP amplicon of WTB (accession number, KP201676.1) and similar sequences of insects present in GenBank. The alignment is displayed here in three sections to better visualize the differences between the WTB sequence (in yellow) and the homologous sequences.