Abstract:Aromia bungii (Faldermann, 1835) (Coleoptera: Cerambycidae), the red-necked longhorn beetle is native to eastern Asia, where it is an important wood-borer of fruit and ornamental species of the genus Prunus. A. bungii is a quarantine pest in the European Union, following its accidental introduction and establishment in Germany and Italy, and is currently included in the list of priority pests. To confirm its infestations in outbreak areas, adult or larval specimens are needed to perform morphological or molecu… Show more
“…Table 5 compares the LoD values obtained in the different techniques. The data assigned to the PCR protocols (probe for hydrolysis and SYBR Green) (Rizzo et al 2020a ), have been omitted in this table.…”
Section: Resultsmentioning
confidence: 99%
“…In some farms situated in the pest outbreak area around Naples (Campania, Italy), where A. bungii is considered as established (Carella 2019 ), frass samples (Fig. 1 ) were collected at the trunk base of Prunus plants and individually labeled as in Rizzo et al ( 2020a ). Fig.…”
Section: Methodsmentioning
confidence: 99%
“…The non-target samples consisted of a set of DNA samples from the entomological biomolecular collection of the phytopathological laboratory of the Phytosanitary Service of the Tuscany Region. The non-target DNA samples were listed in a previous paper (Rizzo et al 2020a ) and included a total of 62 samples belonging to 26 species. They were used for testing the diagnostic specificity of the protocols.…”
Section: Methodsmentioning
confidence: 99%
“…The high specificity and sensitivity of DNA-based technologies allows the detection of harmful organisms even at low concentrations of DNA extracted from plant tissues (Aglietti et al 2019 ; Rizzo et al 2020a ). Among the most versatile, sensitive and specific methods, loop-mediated isothermal amplification (LAMP) can be used as a field-friendly and cost-effective diagnostic tool (Notomi et al 2000 , 2015 ).…”
Section: Introductionmentioning
confidence: 99%
“…This paper presents a reliable and sensitive diagnostic test for the rapid diagnosis of A. bungii frass using the LAMP technique. The quality of this method is compared to the conventional PCR end point method and a qPCR protocol recently developed for the identification of A. bungii from frass (Rizzo et al 2020a ).…”
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.
“…Table 5 compares the LoD values obtained in the different techniques. The data assigned to the PCR protocols (probe for hydrolysis and SYBR Green) (Rizzo et al 2020a ), have been omitted in this table.…”
Section: Resultsmentioning
confidence: 99%
“…In some farms situated in the pest outbreak area around Naples (Campania, Italy), where A. bungii is considered as established (Carella 2019 ), frass samples (Fig. 1 ) were collected at the trunk base of Prunus plants and individually labeled as in Rizzo et al ( 2020a ). Fig.…”
Section: Methodsmentioning
confidence: 99%
“…The non-target samples consisted of a set of DNA samples from the entomological biomolecular collection of the phytopathological laboratory of the Phytosanitary Service of the Tuscany Region. The non-target DNA samples were listed in a previous paper (Rizzo et al 2020a ) and included a total of 62 samples belonging to 26 species. They were used for testing the diagnostic specificity of the protocols.…”
Section: Methodsmentioning
confidence: 99%
“…The high specificity and sensitivity of DNA-based technologies allows the detection of harmful organisms even at low concentrations of DNA extracted from plant tissues (Aglietti et al 2019 ; Rizzo et al 2020a ). Among the most versatile, sensitive and specific methods, loop-mediated isothermal amplification (LAMP) can be used as a field-friendly and cost-effective diagnostic tool (Notomi et al 2000 , 2015 ).…”
Section: Introductionmentioning
confidence: 99%
“…This paper presents a reliable and sensitive diagnostic test for the rapid diagnosis of A. bungii frass using the LAMP technique. The quality of this method is compared to the conventional PCR end point method and a qPCR protocol recently developed for the identification of A. bungii from frass (Rizzo et al 2020a ).…”
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.
Aromia bungii Faldermann (Coleoptera: Cerambycidae) is an emerging invasive pest of economically important Prunus species that is native to China, Mongolia, the Russian Far East, Korea, and Vietnam. It was recently introduced to Japan, Germany, and Italy, where it is spreading and damaging crops and ornamental trees. It exhibits an adaptable lifecycle, a high reproductive output, and the larvae live concealed under the bark of infested trees, which are traits that promote its invasiveness. Detection and monitoring of A. bungii currently rely upon visual identification of infested trees that are usually already damaged, which is inefficient and not target-specific. Current control methods rely primarily upon the labour-intensive physical removal of infested trees. Although native parasitoid natural enemies of A. bungii provide control in Chinese orchards, none are appropriate for classical biological control in invaded areas due to biosafety concerns surrounding their broad host ranges. However, entomopathogenic fungi and nematodes may provide viable options for biological control in invaded ranges. Recent advancements in semiochemical baited traps may provide sustainable, target-specific, and efficacious methods to monitor and control A. bungii. There remains much to learn about the biology and control of A. bungii, and continued advancements in the study of sustainable control tools are needed for the management of this emerging pest.
Molecular assays based on qPCR TaqMan Probes were developed to identify three species of the genus Xylosandrus, X. compactus, X. crassiusculus and X. germanus (Coleoptera Curculionidae Scolytinae). These ambrosia beetles are xylophagous species alien to Europe, causing damages to many ornamental and fruiting trees as well as shrubs. DNA extraction was carried out from adults, larvae and biological samples derived from insect damages on infested plants. For X. compactus, segments of galleries in thin infested twigs were cut and processed; in the case of X. crassiusculus, raw frass extruded from exit holes was used, while DNA of X. germanus was extracted from small wood chips removed around insect exit holes. The assays were inclusive for the target species and exclusive for all the non-target species tested. The LoD was 3.2 pg/µL for the frass of X. crassiusculus and 0.016 ng/µL for the woody matrices of the other two species. Both repeatability and reproducibility were estimated on adults and woody samples, showing very low values ranging between 0.00 and 4.11. Thus, the proposed diagnostic assays resulted to be very efficient also on the woody matrices used for DNA extraction, demonstrating the applicability of the protocol in the absence of dead specimens or living stages.
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