Molecular Identification of Agrilus anxius (Coleoptera: Buprestidae) Using a qPCR Assay With Locked Nucleic Acid (LNA) Probe

Journal of Applied Entomology, Journal Year: 2025, Volume and Issue: unknown

Published: March 6, 2025

ABSTRACT In the present study, a biomolecular diagnostic assay based on qPCR‐LNA (Locked Nucleic Acid) probes was developed for identification of Agrilus anxius (Coleoptera: Buprestidae) from adult insects. The performance new protocol also evaluated indirect diagnosis insect's presence artificially contaminated frass. designed primer and probe were able to distinguish in silico all A. samples nontarget species, with 100% match homologous sequences found GenBank databases. molecular sensitive, specific repeatable. analytical sensitivity (limit detection—LoD) adults frass 6.4 fg/μL 0.08 pg/μL, respectively. This assay, by analysing eDNA samples, will allow early detection an area before it has caused major impact. analysis is becoming increasingly used tool spatial survey programs phytosanitary services could play decisive role pest surveillance.

Language: Английский

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Diagnostic Tool for the Identification of Bactrocera dorsalis (Hendel) (Diptera: Tephritidae) Using Real-Time PCR

Insects, Journal Year: 2024, Volume and Issue: 15(1), P. 44 - 44

Published: Jan. 8, 2024

Accurate identification of Bactrocera dorsalis (Hendel) (Diptera: Tephritidae), commonly known as the Oriental fruit fly, is a significant challenge due to morphological convergence and taxonomic uncertainties species belonging same genus. This highly polyphagous poses threat crops. With its potential establishment in Europe becoming growing concern, there an urgent need for rapid efficient diagnostic methods. The study presented here introduces protocol based on real-time PCR using TaqMan probe early reproducible B. dorsalis. Specimens representing genetic diversity Italian population were collected analyzed. Specific primers designed conserved regions silico analysis confirmed their specificity. assay conditions optimized, analytical sensitivity, specificity, repeatability, reproducibility evaluated. showed high sensitivity accurately detecting low DNA concentrations standardized method provides reliable tool routine diagnostics, enhancing accuracy efficiency identifying fly at all stages development, thereby facilitating effective pest management measures. development this crucial monitoring supporting efforts prevent passive spread Europe, particularly light recent active infestations detected Italy.

Language: Английский

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Streamlining the Identification of the Orange Spiny Whitefly, Aleurocanthus spiniferus (Hemiptera: Aleyrodidae), with Real-Time PCR Probe Technology

Agriculture, Journal Year: 2025, Volume and Issue: 15(4), P. 414 - 414

Published: Feb. 16, 2025

Aleurocanthus spiniferus (Quaintance) (Hemiptera: Aleyrodidae) has rapidly spread, mainly in the central and eastern Mediterranean coastal area, infesting various new host plants alongside known ones. This invasive species poses a significant threat to agricultural ecosystems, necessitating urgent action monitor control outbreaks previously pest-free areas. While entomological morphological recognitions are crucial for initial detection, challenges often arise quickly identifying different developmental stages or genus-level distinctions, particularly surveys conducted by personnel with limited skills. Due these challenges, qPCR probe protocol was developed enhance diagnostic capacity of laboratories responsible territorial pests. biomolecular tool integrates surveys, enabling prompt reliable proof A. presence free areas, delimited territories, during phytosanitary import inspections. The protocol’s high analytical specificity, inclusivity, exclusivity ensure accurate identification spiniferus, while its low limit detection repeatability reproducibility reinforce utility as standardized method. By facilitating targeted efforts, this innovative approach strengthens resilience systems against widespread infestations.

Language: Английский

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

3 Biotech, Journal Year: 2021, Volume and Issue: 11(2)

Published: Jan. 19, 2021

Abstract The red-necked longhorn beetle Aromia bungii (Faldermann, 1835) (Coleoptera: Cerambycidae) is native to east Asia, where it a major pest of cultivated and ornamental species the genus Prunus . Morphological or molecular discrimination adults larval specimens required identify this invasive wood borer. However, recovering stages from trunks branches causes extensive damage plants timewasting. An alternative approach consists in applying non-invasive diagnostic tools biological traces (i.e., fecal pellets, frass). In way, infestations host can be detected without destructive methods. This paper presents protocol based on both real-time visual loop-mediated isothermal amplification (LAMP), using DNA A. extracted particles frass. Laboratory validations demonstrated robustness protocols adopted their reliability was confirmed performing an inter-lab blind panel. LAMP assay qPCR SYBR Green method F3/B3 external primers were equally sensitive, more sensitive than conventional PCR (sensitivity > 10 3 same starting matrix). protocol, due relatively easy performance method, could useful tool apply rapid monitoring management its outbreaks.

Language: Английский

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Identification of the ambrosia beetle Xyleborinus saxesenii (Ratzeburg, 1837) (Coleoptera: Curculionidae: Scolytinae) from frass and adult DNA by TaqMan Probe real‐time‐PCR

EPPO Bulletin, Journal Year: 2024, Volume and Issue: 54(1), P. 84 - 94

Published: Jan. 22, 2024

Abstract A molecular tool has been set up for the unambiguous identification of ambrosia beetle, Xyleborinus saxesenii (Ratzeburg, 1837) (Coleoptera Curculionidae Scolytinae), cosmopolitan fruit‐tree pinhole borer, widely distributed in temperate regions five continents, where it infests a wide range hardwoods and softwoods. The test was based on real‐time PCR with TaqMan probe technology developed whole insect bodies (adults) as well frass produced by beetle. shown to meet criteria established EPPO harmonization diagnostic methods. In particular, gave good results terms analytical specificity (inclusivity exclusivity) sensitivity, fully repeatable reproducible. Since X. is one most commonly intercepted beetles during phytosanitary controls worldwide, this practical will be useful its rapid beetle biological material (frass, body fragments). can countries quarantine species, region.

Language: Английский

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

Plants, Journal Year: 2021, Volume and Issue: 10(6), P. 1048 - 1048

Published: May 22, 2021

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

Language: Английский

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Biology, impact, management and potential distribution of Aromia bungii, a major threat to fruit crops around the world

Journal of Pest Science, Journal Year: 2024, Volume and Issue: 97(4), P. 1725 - 1747

Published: April 15, 2024

Abstract Aromia bungii Faldermann (Coleoptera: Cerambycidae) is an emerging invasive pest of economically important Prunus species that native to China, Mongolia, the Russian Far East, Korea, and Vietnam. It was recently introduced Japan, Germany, Italy, where it spreading damaging crops ornamental trees. exhibits adaptable lifecycle, a high reproductive output, larvae live concealed under bark infested trees, which are traits promote its invasiveness. Detection monitoring A. currently rely upon visual identification trees usually already damaged, inefficient not target-specific. Current control methods primarily labour-intensive physical removal Although parasitoid natural enemies provide in Chinese orchards, none appropriate for classical biological invaded areas due biosafety concerns surrounding their broad host ranges. However, entomopathogenic fungi nematodes may viable options Recent advancements semiochemical baited traps sustainable, target-specific, efficacious monitor . There remains much learn about biology , continued study sustainable tools needed management this pest.

Language: Английский

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A duplex real-time PCR with probe for simultaneous detection of Geosmithia morbida and its vector Pityophthorus juglandis

PLoS ONE, Journal Year: 2020, Volume and Issue: 15(10), P. e0241109 - e0241109

Published: Oct. 23, 2020

The cultivation of walnuts (Juglans sp.) in Europe retains high economic, social, and environmental value. recent reporting the Thousand Cankers Disease (TCD) fungus, Geosmithia morbida, its vector, Pityophthorus juglandis, walnut trees Italy is alarming whole Europe. Although at present only foothold disease outside North America, given difficulties inherent traditional identification both members this beetle/fungus complex, a rapid effective protocol for early detection TCD organisms an absolute priority Here we report development sensitive molecular tool based on simplex/duplex qPCR assays rapid, accurate highly specific bionectriaceous fungal pathogen bark-beetle vector. Our assay performed excellently, detecting minute amounts target DNA without any non-specific amplification. Detection limits from various heterogeneous matrices were lower than other reported assays. could assist organism interception entry points, territory monitoring eradication outbreaks, delineation quarantine areas, tracing back dispersal pathways.

Language: Английский

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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)

3 Biotech, Journal Year: 2021, Volume and Issue: 11(6)

Published: May 10, 2021

Abstract Molecular assays based on qPCR TaqMan Probes were developed to identify three species of the genus Xylosandrus , X. compactus, crassiusculus and germanus (Coleoptera Curculionidae Scolytinae). These ambrosia beetles are xylophagous alien Europe, causing damages many ornamental fruiting trees as well shrubs. DNA extraction was carried out from adults, larvae biological samples derived insect infested plants. For compactus segments galleries in thin twigs cut processed; case raw frass extruded exit holes used, while extracted small wood chips removed around holes. The inclusive for target exclusive all non-target tested. LoD 3.2 pg/µL 0.016 ng/µL woody matrices other two species. Both repeatability reproducibility estimated adults samples, showing very low values ranging between 0.00 4.11. Thus, proposed diagnostic resulted be efficient also used extraction, demonstrating applicability protocol absence dead specimens or living stages.

Language: Английский

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Rapid identification of Ips sexdentatus (Boerner, 1766) (Curculionidae) from adults and frass with real-time PCR based on probe technology

Journal of Plant Diseases and Protection, Journal Year: 2024, Volume and Issue: 131(5), P. 1473 - 1481

Published: April 27, 2024

Abstract A molecular tool has been developed for the identification of Ips sexdentatus (Börner 1776) (Coleoptera Curculionidae Scolytidae), well-known six thooted bark beetle, widely distributed in Eurasia, where it infests several species genus Pinus and occasionally a few conifer genera Abies , Larix Picea . The test can be useful both countries I. is handled as quarantine and, to greater reason, Europe discriminate biological traces this commonly found beetle from those produced by regulated pests. protocol based on real-time PCR with TaqMan probe technology whole insect bodies (adults) well artificial frass contaminated DNA beetle. here direct indirect proven effective terms analytical specificity, sensitivity, reliability reproducibility. recommended practical diagnostic allowing rapid toothed presence any trace other xylophagous pests collected at points entry during phytosanitary surveys.

Language: Английский

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