Development of a loop-mediated isothermal amplification (LAMP) assay for the identification of the invasive wood borer Aromia bungii (Coleoptera: Cerambycidae) from frass

Abstract

The red-necked longhorn beetle Aromia bungii (Faldermann, 1835) (Coleoptera: Cerambycidae) is native to east Asia, where it is a major pest of cultivated and ornamental species of the genus Prunus. Morphological or molecular discrimination of adults or larval specimens is required to identify this invasive wood borer. However, recovering larval stages of the pest from trunks and branches causes extensive damage to plants and is timewasting. An alternative approach consists in applying non-invasive molecular diagnostic tools to biological traces (i.e., fecal pellets, frass). In this way, infestations in host plants can be detected without destructive methods. This paper presents a protocol based on both real-time and visual loop-mediated isothermal amplification (LAMP), using DNA of A. bungii extracted from fecal particles in larval frass. Laboratory validations demonstrated the robustness of the protocols adopted and their reliability was confirmed performing an inter-lab blind panel. The LAMP assay and the qPCR SYBR Green method using the F3/B3 LAMP external primers were equally sensitive, and both were more sensitive than the conventional PCR (sensitivity > 103 to the same starting matrix). The visual LAMP protocol, due to the relatively easy performance of the method, could be a useful tool to apply in rapid monitoring of A. bungii and in the management of its outbreaks.

TaqMan probe assays on different biological samples for the identification of three ambrosia beetle species, Xylosandrus compactus (Eichoff), X. crassiusculus (Motschulsky) and X. germanus (Blandford) (Coleoptera Curculionidae Scolytinae)

“…In recent years, many biomolecular diagnostic tools able to discriminate the presence and to identify insect pests were developed: termites (Ide et al 2016a ), bugs and cicadids (Bouwer et al 2014 ; Bracalini et al 2015 ), moths (Onah et al 2016 ; Kang et al 2019 ), wood-boring beetles (Ide et al 2016b ; Cognato et al 2020 ; Rizzo et al 2021 ). The biomolecular identification methods can be efficient and allow a prompt implementation of control strategies (EFSA 2020 ) for the detection and eradication of quarantine pests (Augustin et al 2012 ; Landi et al 2017 ; Rizzo et al 2020a ).…”

Molecular identification of Anoplophora glabripennis (Coleoptera: Cerambycidae) and detection from frass samples based on real-time quantitative PCR

“…Strangi proposed a protocol of molecular analyses based on end point polymerase chain reaction (PCR) amplification of DNA samples extracted from A. chinensis frass collected from potentially infested host trees (Strangi et al 2013). Rizzo and colleagues provided diagnostic tests for the detection of Aromia bungii from frass samples based on real-time quantitative PCR (qPCR) (Rizzo et al 2020a) and Loop-mediated Isothermal Amplification (LAMP) (Rizzo et al 2021). A diagnostic protocol based on LAMP technology enabling the rapid detection of A. glabripennis DNA from frass samples was developed and validated (Rizzo et al 2020b).…”

The Potential Use of Isothermal Amplification Assays for In-Field Diagnostics of Plant Pathogens

“…An increment borer enables wood samples to be obtained from living trees which can be homogenized by grinders or mortars [37,38]. Non-invasive collection of samples (wood chips or frass) can also be used for subsequent grinding and DNA extraction [39,40]. Therefore, various convenient tools are available and can be used for pre-treating different plant tissues in the field.…”