Novel Agrilus planipennis early-detection tool designed on Cytochrome B gene

Novel Agrilus planipennis early-detection tool designed on Cytochrome B gene | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Novel Agrilus planipennis early-detection tool designed on Cytochrome B gene Domenico Rizzo, Alessandra Gionni, Francesco Pecori, Claudia Gabriela Zubieta, and 13 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7980969/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 6 You are reading this latest preprint version Abstract Agrilus planipennis is a quarantine pest that is threatening native ash populations in both North America and Europe. Early detection of this pest, which can cause severe infestations, is essential, and molecular tests applied at different developmental stages and environmental traces can contribute substantially to improving control measures to prevent or contain A. planipennis infestations. In this study, a specific A. planipennis real-time qPCR assay was developed using a Locked Nucleic Acid (LNA) probe based on the CytB (Cytochrome B) gene. The detection limit of this method was 25.6 fg/µl for adult DNA extracts and 0.21 pg/µl for frass produced by A. planipennis larvae. The new qPCR probe test, which targets a different locus, not only allows identification of the pest and provides an indirect diagnosis through environmental DNA analysis but can also be used for cross-validation of results between different tests. emerald ash borer quarantine pest early detection molecular diagnostic tool phytosanitary surveillance Figures Figure 1 Figure 2 Figure 3 Figure 4 1 Introduction The Emerald Ash Borer (EAB), Agrilus planipennis Fairmaire (Coleoptera: Buprestidae), is a xylophagous buprestid, primary associated with trees of the genus Fraxinus . All European ash species are susceptible hosts of this beetle. This species is native to Asia (China, Japan, Korea, Mongolia and the Russian Far East) and has been accidentally introduced into North America and Western Russia, from where it has spread to Ukraine (Haak et., 2002, Liu et al., 2003 , Baranchikov et., 2008, Drogvalenko et al., 2019 , Evans et., 2020) and most recently to Belarus (Zviagintsev et al., 2025 ). Agrilus planipennis is a quarantine pest listed in Part A of Annex II of Regulation (EU) 2019/2072. In addition, the European Commission carried out a risk assessment of these pests and identified 20 priority pests whose introduction can cause particularly high economic, ecological and social damage, including A. planipennis (Regulation (EU) 2019/1702). A recent EFSA report on the potential impact of its introduction into the EU, estimated that 87.6% of ash trees could be lost, based on the considered scenario assumptions (EFSA, 2025). Heavy infestations of A. planipennis cause symptoms, such as smaller or discoloured leaves, progressive crown dieback, formation of epicormic shoots on branches and stems, bark cracking and flaking due to predation by woodpeckers and even the plant’s death. Furthermore, attacked trees show D-shaped insect exit holes of about 3–4 mm, serpentine larval galleries under the bark, detached bark and debarking. However, tree decay due to insect infestation can only be detected after years, when the population density has increased significantly. Indeed, A. planipennis often remains undetected for several years in new environments (EFSA, 2024). These considerations suggest that surveillance, based on visual examination and sampling of plant parts, is not sufficient for the early detection of new EAB outbreaks. Recent experiences in North America indicate that trapping is the most effective method for EAB detection (EFSA, 2024). However, it must be considered that, in general, the morphological identification of Agrilus species appears to be challenging (EPPO Global Database), especially due to the size of the genus, comprising more than 3000 species (Volkovitsh et al., 2020 ). Molecular identification of insects is a rising method, especially for quarantine species, for which rapid identification is crucial. In the case of xylophagous insects, the most frequently examined traces are frass samples in feeding tunnels or around entry/exit holes dug into trunks and branches (Rizzo et al., 2020, EPPO, 2021 ). DNA shed by organisms into environment samples is considered environmental DNA (eDNA). Molecular approaches based on eDNA offer several advantages: rapid detection via direct gene amplification, reduced reliance on taxonomic expertise, and minimal disturbance to the surveyed host. Numerous diagnostic protocols exploiting frass samples or other genetic residues have been developed in recent years (Ide et al., 2016 , Kyei-Poku et al., 2020 , Rizzo et al., 2020, Kyle et al., 2024 ). Specific assays, targeting COI gene, to identify A. planipennis and detect traces of its DNA within frass samples, were developed and validated on other European wood-borer species (Peterson et al. 2023, Kupper et al., 2025). In this study, we developed a new sensitive real-time qPCR assay, based on an alternative locus (Cytochrome B), with a Locked Nucleic Acid (LNA) probe for A. planipennis detection. LNA probes and primers contain chemically modified bases that increase the rigidity of the ribose. This modification improves the stability, specificity, and affinity for base-pairing with target sequences (Josefsen et al., 2009). The high binding affinity allows for shorter probes, which results in higher binding specificity to the target DNA. The assay was validated using adult insect tissue and synthetic frass matrices, demonstrating its suitability for routine laboratory diagnostic applications. 2 Materials and methods 2.1 Samples Target and non-target organisms (including other genera present on the same host plants) were obtained from different sources and geographical origins (Table 1). The target specimens include 8 A. planipennis adults and 6 artificial frass samples (obtained according to Rizzo et al., 2025). Among the 131 outgroup samples, 55 belong to Agrilus spp. and 76 to other genera. To assess the efficiency and specificity of the assay a GStrand (In 338) synthetic DNA (Eurofins Genomics), equivalent to the expected amplicon and based on the CytB gene of A. planipennis (Fig. 1 ) was synthesised. All sample details are listed in Table 1. Table 1. Samples from insects and frass used in this study. Species Sample code Life stage/sample type Supplier b Agrilus planipennis* MR 000814 Adult USDA MR 001563 MR 001713 MR 001638 MR 001714 MR 001714/2 MR 001714/3 MR 001714/4 MR_Frass_Ap Artificial Frass PPS-T MR_Frass_Ap/1 MR_Frass_Ap/2 MR_Frass_Ap/3 MR_Frass_Ap/4 MR_Frass_Ap/5 In 338 gStrand PPS-T Agrilus angustulus* MR 001710 Adult JKI MR 001721/IIC Agrilus anxius* MR 001715 Adult JKI MR 001716 MR 001717 MR 001718 MR 001719 MR 001720/(BBB11) † RU MR 001721/(BBB12) † MR 001722/(BBB20) † GLFC MR 001723 Artificial Frass JKI MR 001724 MR 001725 MR 001726 Agrilus auroguttatus* MR 001701 Adult UCa MR 001689 MR 001689/2 MR 001689/3 MR 001689/4 MR 001689/5 MR 001689/6 MR 001689/7 MR 001689/F Frass UCa MR_Frass_Au Artificial Frass PPS-T MR_Frass_Au/1 MR 001689/Fc Fecal residue UCa Agrilus biguttatus MR 001722/IIC Adult JKI MR 001524 PPS-T Agrilus convexicollis MR 001712 Adult JKI MR 001720/IIC Agrilus graminis* In 308 † Adult INRAE Agrilus hastulifer In 309 † Adult INRAE Agrilus laticornis* MR 001709 Adult JKI MR 001718 Agrilus obscuricollis In 310 † Adult INRAE Agrilus olivicolor MR 001716 Adult JKI MR 001717/1 UoF Agrilus sulcicollis MR 001717 Adult JKI Agrilus viridis fagi MR 001711 Adult UoF MR 001719 JKI Anoplophora chinensis* MR 001617 Adult PPS-T MR 001618 MR 001648 MR 001620 MR 000225 Larva PPS-L MR 000259 PPS-T MR 001619 MR 001678 MR 001622 MR 001649 MR 000790 Frass PPS-T MR 001621 MR 001624 MR 001639 MR 001640 MR 001668 Anoplophora glabripennis* MR 000226 Adult PPS-L MR 000757 Larva MR 000301 Frass MR 001625 Araecerus fasciculatus* MR 000824 Larva PPS-T_PLI Aromia bungii* MR 000260 Adult PPS-C MR 000261 MR 000262 MR 001674 MR 000254 Larva MR 000231 Frass PPS-C MR 000263 UoN MR 001688 Aromia moschata MR 000766 Adult Buprestis cupressi MR 001707 Adult UoF Cerambyx cerdo MR 000274 Adult CREA_DC MR 000297 Cerambyx scopolii MR 000275 Adult CREA_DC MR 000756 Cerambyx welensii MR 000755 Adult CREA_DC Cerambyx miles MR 000829 Adult CREA_DC Chlorophorus glabromaculatus MR 000799 Larva PPS-T_PLI Chrysobothris affinis* MR 001944 Larva PPS-T_PLI Chrysobothris igniventris* MR 001954 Larva PPS-T_PLI Chrysobothris quadriimpressa* MR 000853 Larva PPS-T_PLI MR 001694 Chrysobothris rugosiceps* MR 001699 Larva PPS-T_PLI MR 001700 Chrysobothris solieri* MR 001702 Larva PPS-T_PLI Chrysobotris femorata* MR 001615 Larva PPS-T_PLI Cossus cossus MR 000831 Adult UoP MR 000265 Larva PPS-T MR 000289 Frass PPS-T Graphisurus fasciatus* MR 000809 Larva PPS-T_PLI MR 001695 Hylurgus ligniperda MR 000302 Adult UoF Hylurgopinus rufipes MR 000855 Larva PPS-T_PLI Larinus planus MR 001923 Larva PPS-T_PLI Leptostylus asperatus* MR 001948 Larva PPS-T_PLI Lepturges confluens* MR 001692 Larva PPS-T_PLI Monochamus galloprovincialis MR 000250 Adult CREA_DC MR 000258 Adult PPS-L MR 000818 Adult UoF Monochamus sartor MR 000257 Adult PPS-L MR 000256 Larva Morimus asper MR 000759 Adult CREA_DC Neoclytus acuminatus* MR 001704 Adult UoF Neoclytus mucronatus* MR 000852 Larva PPS-T_PLI Phryneta leprosa* MR 000828 Adult PPS-T_PLI MR 000828/L Larva Saperda carcharias MR 000767 Adult UoP Saperda punctata* MR 000784 Adult CREA_DC Saperda scalaris* MR 000785 Adult CREA_DC Saperda tridentata* MR 000294 Adult PPS-T_PLI MR 001690 Adult MR 001691 Adult MR 000247 Larva Stictoleptura cordigera* MR 000792 Adult UoF Zeuzera pyrina MR 000837 Larva UoP MR 000838 Frass * Identified by sequence analysis of COI gene, using primers LCO1490/HCO2198 (Folmer et al., 1994). Provided as DNA sample. b UoF = University of Florence, Italy; JKI = Julius Kühn Institute, Germany; UoP = University of Pisa, Italy; USDA = U.S. Department of Agriculture APHIS PPQ, Brighton, MI; UCa = University of California, USA; UoN = University of Naples, Italy; PPS-T = Plant Protection Service of Tuscany, Italy; PPS-T_PLI = Plant Protection Service of Tuscany, Port of Leghorn interception, Italy; CREA_DC = Consiglio per la Ricerca in agricoltura e l’analisi dell’Economia Agraria, Italy; PPS-C = Plant Protection Service_Campania, Italy; PPS-L = Plant Protection Service_Lombardy, Italy; RU = Rutgers University, USA; GLFC = Great Lakes Forestry Centre, Canada; INRAE = Laboratoire de Biologie des Ligneux et des Grandes Cultures, France. 2.2 DNA extraction DNA extraction was performed using a 2% CTAB buffer, followed by purification with Maxwell® RSC PureFood GMO and Authentication Kit (Promega Italia, Milan, Italy) (Rizzo et al., 2023). Artificial frass of EAB was produced by mixing CTAB lysates obtained from Fraxinus excelsior wood chips and from A. planipennis adult specimen (sample MR001638 – Table 1). Concentration and quality of the extracted DNA were quantified by using Qiaexpert (Qiagen). DNA obtained from both, target and non-target organisms (Table 1) was normalized to a concentration of 5 ng/µl and used for qPCR reactions and stored at -20°C until use. DNA amplifiability was verified by qPCR reactions using a TaqMan probe based on 18S rDNA region (Ioos et al., 2009 ) as described in Rizzo et al. (2025). 2.3 Primers and probe design for real-time PCR assay To identify the specific genomic region suitable for oligonucleotide design, several sequences were analysed and evaluated. Their selection was based on the following factors: presence of polymorphisms, possibility of comparing similar sequences from other congeneric or genetically related species, use of specific mitochondrial genomic regions (Fig. 1 ) and highly conserved genes that provide interspecific variability (Kralik et al., 2017). Based on the previous considerations and sequence availability, the CytB locus was selected. CytB gene is characterized by low intraspecific variability (genetic distance 8%), allowing the distinction between cryptic or morphologically similar insect species (Chen et al., 2023). Primers and probe were designed, using the online software OligoArchitec primer design (Sigma-Aldrich, St. Louis, USA), on the CytB region of A. planipennis (KT363854.1) obtained from The National Center for Biotechnology Information ( https://www.ncbi.nlm.nih.gov ). The primers and LNA probe were synthesized by Eurofins Genomics (Ebersberg, Germany) (Fig. 2). To evaluate the in silico specificity, a total of 640 sequences from 103 different Agrilus species were aligned and compared with the newly designed amplicon (Figure S1 , supplementary material) by using the Geneious software. These sequences belong to species with the greatest genetic affinity, similar ecological niches or the potential for morphological misidentification (Volkovitsh et al., 2020 ). The amplicon specificity was verified by using the BLAST software (Basic Local Alignment Search Tool; http://www.ncbi.nlm.nih.gov/BLAST ) and a partial sequences alignment is shown in Fig. 3 . 2.4 Optimisation and validation of the qPCR method Based on the melting temperatures of the oligos, the real-time PCR probe protocol has been optimized for the annealing temperature by preparing a gradient (between 52°C and 62°C). The oligo concentration was evaluated as follows: 0.1 µM, 0.2 µM, 0.3 µM, and 0.4 µM for the primers and probe. For each run, two tubes containing nuclease-free distilled water (no template control - NTC), a positive and a negative amplification control were tested. Validation was performed according to the EPPO Standard PM7/98 (5). The specificity of the qPCR assay was tested for all samples listed in Table 1 by normalising their DNA concentration to 5 ng/µL. The limit of detection (LoD) was determined on 1:5 serial dilutions of target DNA extracts ( A. planipennis adult) ranging between 10 ng/µL to 5.12 fg/µL. Each dilution point was analysed in triplicate. The standard curve generated from the same serial dilution was used to quantify the target DNA in the artificial frass. Intra-variation (repeatability) and inter-variation (reproducibility) were performed and evaluated based on mean Cq values and their standard deviations (Dhami et al., 2016 , Groth-Helms et al., 2023). Repeatability and reproducibility were performed using DNA extracts from A. planipennis adult, normalized to a concentration of 16 pg/µL. 2.5 Blind test The blind test was carried out with 12 blind samples, at different DNA concentrations (10, 2 and 0.5 ng/µL): 6x A. planipennis , 2x A. anxius , 2x A. sulcicollis , 1x A. graminis , 1x Negative Template control (NTC). Each sample was numbered progressively and processed in triplicates. The laboratories involved in the blind panel were: 1) LAB 1 (organizer): Phytopathological laboratory of the Tuscany Regional Phytosanitary Service, headquarters of Pistoia (Italy); 2) LAB 2: Phyto pathological laboratory of the Tuscany Regional Phytosanitary Service, headquarters of Livorno (Italy); 3) LAB 3: Phytopathological laboratory of the Tuscany Regional Phytosanitary Service, headquarters of Florence (Italy); LAB 4: laboratory of the CNR- IPSP of Sesto Fiorentino—Florence (Italy). Each laboratory conducted the qPCR analyses using the same diagnostic protocol developed in this study, but with different instruments and master mixes: i) LAB 1_ CFX96 (Bio-Rad) thermocycler and Quantinova Probe (Qiagen) master mix; ii) LAB 2: ARIA MMX (Agilent) thermocycler and Luna universal Probe (NEB) Master mix; iii) LAB. 3: Line Gene 96 Plus (Bioer, Rome, Italy) thermocycler and Quantinova Probe (Qiagen) master mix; iv) LAB 4: Rotor-Gene Q (Qiagen) thermocycler and TaqMan Universal PCR Master Mix (Applied Biosystems). The evaluation of the performance of the participating laboratories was assessed by considering the qualitative values (Positive/Negative) recorded in the PT results sheet (Chabirand et al., 2014 ). The results were interpreted for each laboratory by calculating the number of true positives (TP), true negatives (TN), false positives (FP) and false negatives (FN), according to the Table 2 . Table 2 Parameters considered in the blind panel: sensitivity, specificity and diagnostic accuracy. Performance Criteria Definition Equation Diagnostic sensitivity Concordance between the analysis result and the assigned value of the samples, for which a POSITIVE value was assigned TP/(TP + FN) x 100% Diagnostic specificity Concordance between the analysis result and the assigned value of the samples, for which a NEGATIVE value was assigned TN/(TN + FP) x 100% Accuracy Concordance between analysis result and assigned value (TP + TN)/N x 100% TP: True positives; TN: True negatives; FP: False positives; FN: False negatives; N: Total number of samples. 3 Results 3.1 Assay conditions of the qPCR LNA Probe protocol Real-time amplification reactions with the LNA probe were optimised considering the reaction mixture, primer annealing temperatures and optimal primer concentrations. The optimal reaction mixture consisted of the following reagents, in a final volume of 20 µL: 10 µL of QuantiNova Supermix 2x (Qiagen, Hercules, USA), with primer concentrations at 0,4 µM, 0,2 µM for LNA probe and 2 µL of DNA template. As for the optimal thermal protocol, the conditions are as follows: initial denaturation at 95°C for 2 minutes, followed by 40 cycles of 95°C for 15 s and annealing at 60°C for 40 s. 3.2 DNA extraction Quality and amplifiability of extracted DNAs are shown in Table 3 . The Cq values were obtained by real-time PCR amplifying the ribosomal 18S gene (Ioos et al., 2009 ). A total of eight adult specimens of A. planipennis and six samples of artificial frass were evaluated. Table 3 Performance of DNA extraction from adult Agrilus planipennis and artificial frass, based on mean concentrations of extracted DNA (± SD), absorbance ratio (A260/280) and mean Cq values of 18S (Ioos et al., 2009 ). Sample Mean DNA Concentration (ng/µL) Absorbance (A260/280) Mean Cq (18S) Adult (n = 8) 73.53 ± 15.07 1.77 ± 0.06 18.2 ± 0.24 Artificial frass (n = 6) 49.22 ± 2.72 1.94 ± 0.08 24.2 ± 0.34 3.3 qPCR assay validation NCBI BLAST ® results showed a high specificity of the qPCR amplicon with A. planipennis sequences (Identity = 100%; E-value = 1e-75); no relevant homology was found with any closely related species. The qPCR assay developed in this study provided comprehensive and exclusive results for A. planipennis with a clear non-specificity towards the non-target organisms. The Limit of Detection (LoD) from A. planipennis DNA adult was 25.6 fg/µL (Cq mean ± SD = 36.48 ± 0.28) (Table 4 ). Table 4 – Agrilus planipennis analytical sensitivity assays using 1:5 serial dilutions (from 10 ng/µL to 5.12 fg/µL) in triplicate (A, B, and C). The Cq values are the mean of the three threshold cycles of each dilution. Concentration values A B C Average Cq SD (±) 10 ng/µL 18.27 18.42 18.38 18.36 0.06 2 ng/µL 20.72 20.73 20.75 20.73 0.01 0.4 ng/µL 23.07 23.15 23.11 23.11 0.03 0.08 ng/µL 25.4 25.35 25.49 25.41 0.06 0.0016 ng/µL 27.53 27.61 27.56 27.57 0.03 3.2 pg/µL 30.38 30.26 30.06 30.23 0.13 0.64 pg/µL 32.2 32.22 32.11 32.18 0.05 0.13 pg/µL 34.94 34.94 34.87 34.92 0.03 25.6 fg/µL 36.33 36.87 36.23 36.48 0.28 5.12 fg/µL n/a n/a n/a n/a n/a The standard curve generated from 1:5 serial dilution A. planipennis DNA showed the following parameters: correlation coefficient (R 2 ): 0.999; slope: -3.3; Y-intercept: 21.7. The qPCR assay showed a 100% of efficiency (E) (Fig. 4 ). The qPCR performed on A. planipennis adult DNA revealed a repeatability and reproducibility of 100% (Table 5 ). Table 5 Repeatability and reproducibility values indicating replicates and corresponding mean (Cq ± SD values) from A. planipennis adult DNA (normalized at 16 pg/µL concentration). Repeatability Reproducibility Replicates Test A Test B Test C Test A Test B Test C 1 28.24 27.9 27.91 28.1 27.85 28.04 2 28.06 27.75 28.27 27.74 27.81 28.03 3 27.72 27.87 27.97 27.83 27.92 27.92 4 27.7 27.77 28.03 28.26 28.75 28.1 5 28 28.52 27.8 28.07 29.14 28.4 6 28.57 28.16 27.87 27.94 28.06 28.15 7 28.51 28.08 28.45 28.95 28.77 28.63 8 28.72 29.03 27.76 28.5 28.11 28.66 Cq Mean 28.19 28.14 28.01 28.17 28.30 28.24 S.D. 0.36 0.41 0.22 0.37 0.48 0.27 DNA extracts from artificial A. planipennis frass were diluted 1:10 up to the eighth dilution. Agrilus planipennis DNA was detected up to the fifth dilution (Cq average = 33.16 ± 0.17), that corresponds to 0.21 ± 0.03 pg/µL (this value was found by interpolating the data from a standard curve using CFX Maestro 1.3 software). This LoD corresponds to the minimum amount of A. planipennis DNA detectable indirectly via frass. 3.4 Blind panel test The blind panel test performed in 4 different laboratories showed a diagnostic sensitivity, specificity and accuracy of 100%. No false positives or false negatives were obtained (Table 6). Table 6. Results of blind panel test from 4 different laboratories by using the qPCR probe LNA assay to detect A. planipennis. Sample Code Species DNA Concentration Expected result LAB 1 LAB 2 LAB 3 LAB4 Ap1 Agrilus planipennis 10 ng/µL Positive + + + + Ap 2 Agrilus planipennis 2 ng/µL Positive + + + + Ap 3 Agrilus graminis 10 ng/µL Negative - - - - Ap 4 Agrilus anxius 10 ng/µL Negative - - - - Ap 5 Agrilus planipennis 0.5 ng/µL Positive + + + + Ap 6 Agrilus anxius 10 ng/µL Negative - - - - Ap 7 Agrilus sulcicollis 10 ng/µL Negative - - - - Ap 8 Agrilus planipennis 10 ng/µL Positive + + + + Ap 9 Agrilus sulcicollis 10 ng/µL Negative - - - - Ap 10 Agrilus planipennis 2 ng/µL Positive + + + + Ap 11 Agrilus planipennis 5 ng/µL Positive + + + + Ap 12 NTC - Negative - - - - LAB 1: CFX96 (Bio-Rad) thermocycler and Quantinova Probe (Qiagen) master mix; LAB 2: ARIA MMX (Agilent) thermocycler and Luna universal Probe (NEB) master mix; LAB. 3: Line Gene 96 Plus (Bioer, Rome, Italy) thermocycler and Quantinova Probe (Qiagen) master mix; LAB 4: Rotor-Gene Q (Qiagen) thermocycler and TaqMan Universal PCR Master Mix (Applied Biosystems). 4 Discussion The presence of A. planipennis poses a serious threat to ash forests and ecosystems in which these plants play a key role. Prevention, quarantine measures, surveillance, and early detection are essential to reduce its impact and limit its spread. Challenges related to morphological identification and detection at low population densities require the development of rapid, sensitive molecular tools for detecting traces of target DNA. The mitochondrial DNA genes cytochrome B (CytB) and cytochrome C oxidase subunit I (COI) are both used as barcode markers for insects (Ray et al., 2024). To date, molecular methods for detecting EAB have been designed based on cytochrome C oxidase subunit I (COI) (Kyei-Puku et al., 2020, Kyle et al., 2024 , Kupper et al., 2025). To reduce the risk associated with non-specific amplification in eDNA analysis and make the results more reliable, it may be helpful to use a combination of assays developed on different gene regions. In this study, we developed an alternative qPCR assay using LNA based on another region of mitochondrial DNA, cytochrome B (CytB). The CytB genomic region provides sufficient variability to allow unequivocal diagnosis of the target species; the use of an LNA probe yielded results comparable to those obtained for the detection and identification of A. anxius (Rizzo et al., 2025). The high specificity of the in silico analysis results was confirmed by in vivo experiments on target and non-target species. The sensitivity of the proposed qPCR protocol (LoD of 25.6 fg/µL with an average Cq of 36.48 ± 0.28) is comparable to that obtained with the LAMP test (20 fg/µL) (Peterson et al., 2023) on adult insects. This comparison with LAMP highlights the qPCR assay as a valid complementary molecular diagnostic tool. Given their distinct methodologies, both approaches are crucial for diagnostic confirmation and data reliability. Similar to other protocols for the indirect diagnosis of pests of interest, the developed protocol was able to detect genetic traces of the target organism in the environment (Kiewnick et al., 2015 , Rizzo et al., 2024). For diagnosis based on frass produced by A. planipennis larvae, the detection limit was 0.21 ± 0.03 pg/µL. These values ensure that the indirect approach is reliable in verifying the presence of A. planipennis in a given area. The robustness of the method was validated through proficiency tests conducted in four independent laboratories. In the context of national surveys or containment and/or eradication measures, to prevent the establishment and spread of Agrilus planipennis within the EU (Regulation (EU) 2024/434), rapid and reliable identification is needed. For these reasons this method could be valuable at cross-border entry points and in high-risk areas (e.g. nurseries, parks), particularly within the European Union, which continues to face a significant risk of EAB introduction and spread. Declarations Author Contributions: Conceptualization of the research approach, detailed laboratory methodologies, and experimental designs for this study were developed and conducted by D. Rizzo (principal investigator) with the assistance of C. G. Zubieta, A. Marrucci, B. Palmigiano, M. Moriconi, C. Ranaldi, N. Luchi, A. Santini, F. Pecori, L. Bartolini, M. Bracalini, B. Hoppe, S. Feltgen, and T. Panzavolta. All sample preparations, data collection, and statistical analyses of data collected from DNA extracts were completed by D. Rizzo, C. G. Zubieta, M. Moriconi, B. Palmigiano, and A. Marrucci. Data curation and data mining, reference assembly, and manuscript formatting were done by D. Rizzo, A. Gionni, N. Luchi, F. Pecori, A. Santini, S. Feltgen, T. Panzavolta, M. Bracalini and B. Hoppe. Writing of the original draft was prepared by D. Rizzo, A. Gionni, F. Pecori, A. Santini, N. Luchi, T. Panzavolta, M. Bracalini, S. Feltgen and B. Hoppe. Revisions of manuscripts were completed by all authors. ACKNOWLEDGEMENTS The authors are grateful to Dr. Benjamin Slager, supervising entomologist at the USDA APHIS PPQ, for providing the target insect specimens needed to set up the method. Conflicts of Interest The authors declare no conflict of interest. References Baranchikov Y, Mozolevskaya E, Yurchenko G, Kenis M (2008) Occurrence of the emerald ash borer, Agrilus planipennis in Russia and its potential impact on European forestry. EPPO Bull 38:233–238. https://doi.org/10.1111/j.1365-2338.2008.01234.x Chabirand A, Anthoine G, Pierson O, Hostachy B (2014) The organization of proficiency testing in plant pathology according to the ISO/IEC 17043: example of the French national reference laboratory. 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Great Lakes Entomol 36:191–204 Peterson DL, Kyle K, Sallé A, Pecori F, Migliorini D, Santini A et al 2023 Specificity and sensitivity of a rapid LAMP assay for early detection of emerald ash borer (Agrilus planipennis) in Europe Forests 14, 436. https://doi.org/10.3390/f14020436 Ray PP, Barala B, Dash P 2024 Cytochrome b gene as a potential DNA barcoding marker in ecoraces of tropical Tasar silkworm Antheraea mylitta. Drury Gene Rep 35, 101922. https://doi.org/10.1016/j.genrep.2024.101922 Rizzo D, Carli M, Zubieta CG, Marrucci A, Ranaldi C, Palmigiano B et al 2024 When frass meets gold: development of a TaqMan probe-based qPCR for identifying goldspotted oak borer, Agrilus auroguttatus Schäffer Unpublished manuscript Rizzo D, Pecori F, Moriconi M, Zubieta CG, Palmigiano B, Bartolini L et al 2025 Molecular identification of Agrilus anxius (Coleoptera: Buprestidae) using a qPCR assay with locked nucleic acid (LNA) probe. J Appl Entomol 149, 757–768. https://doi.org/10.1111/jen.13423 Rizzo D, Taddei A, Da Lio D, Bruscoli T, Cappellini G, Bartolini L et al 2020 Molecular identification of Anoplophora glabripennis (Coleoptera: Cerambycidae) from frass by loop-mediated isothermal amplification. J Econ Entomol 113, 2911–2919. https://doi.org/10.1093/jee/toaa206 Volkovitsh MG, Orlova-Bienkowskaja MJ, Kovalev AV, Bienkowski AO (2020) An illustrated guide to distinguish emerald ash borer (Agrilus planipennis) from its congeners in Europe Forestry. 93:316–325. https://doi.org/10.1093/forestry/cpz024 Zviagintsev VB, Kirichenko NI, Chernik MI, Seraya LG, Baranchikov YN (2025) The Emerald ash borer Agrilus planipennis Fairmaire (Coleoptera: Buprestidae) invaded Belarus. Acta Biologica Sibirica 11:847–861. https://doi.org/10.5281/zenodo.16744135 Supplementary Files FigureS1.png Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Minor revisions 02 Mar, 2026 Reviewers agreed at journal 21 Jan, 2026 Reviewers invited by journal 07 Nov, 2025 Editor invited by journal 31 Oct, 2025 Editor assigned by journal 31 Oct, 2025 First submitted to journal 29 Oct, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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1","display":"","copyAsset":false,"role":"figure","size":262625,"visible":true,"origin":"","legend":"\u003cp\u003ePosition of the developed assay sequences with evidence of the different positions of similar qPCR diagnostic assay (Kyle et al., 2024) or with different techniques (LAMP) (Kyei-Poku et al., 2020). Image produced using Geneious Prime 2025.1.3 software.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7980969/v1/2ca751c3e7d1a2da939509a0.png"},{"id":96252647,"identity":"58cc760e-35d2-4f1d-8957-ce649b32f20d","added_by":"auto","created_at":"2025-11-19 07:41:19","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":646678,"visible":true,"origin":"","legend":"\u003cp\u003ePrimers (blue and red) and LNA probe (purple) designed on target CytB gene for \u003cem\u003eA. planipennis \u003c/em\u003e(accession no. KT363854.1). The area highlighted in grey is equivalent to the specific gStrand (In 338).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7980969/v1/e5821d0fd3e88d4d5910a666.png"},{"id":96250487,"identity":"808ea4ec-0e0e-45db-9781-1b4c3b0e6f4e","added_by":"auto","created_at":"2025-11-19 07:38:27","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":100637,"visible":true,"origin":"","legend":"\u003cp\u003eAlignments between the \u003cem\u003eAgrilus planipennis\u003c/em\u003e amplicon and different sequences of other \u003cem\u003eAgrilus\u003c/em\u003e species. Primers are indicated in green and LNA probe in red.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7980969/v1/1013cbb1a16ab268a7145547.png"},{"id":96170471,"identity":"96644436-5678-443f-a02c-2b4950630a3e","added_by":"auto","created_at":"2025-11-18 10:27:42","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":226646,"visible":true,"origin":"","legend":"\u003cp\u003eAmplification curves (A) and standard curves (B) relating to the qPCR probe assay using 1:5 serial dilutions of \u003cem\u003eA. planipennis \u003c/em\u003einsect adult DNA ranged between 10 ng/µL to 5.12 fg/µL.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7980969/v1/08a2af33990a6f2c71cd77d7.png"},{"id":96256882,"identity":"3435e23e-38e0-4b73-b708-e2d830c9fbdf","added_by":"auto","created_at":"2025-11-19 07:50:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2170277,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7980969/v1/82140ea0-4885-47d2-bac8-f0750dba9746.pdf"},{"id":96170480,"identity":"a2521d16-4b53-45f4-a464-1694d886e6ae","added_by":"auto","created_at":"2025-11-18 10:27:42","extension":"png","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":3241977,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS1.png","url":"https://assets-eu.researchsquare.com/files/rs-7980969/v1/f164f87c0149a13ac8ae36c9.png"}],"financialInterests":"","formattedTitle":"Novel Agrilus planipennis early-detection tool designed on Cytochrome B gene","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eThe Emerald Ash Borer (EAB), \u003cem\u003eAgrilus planipennis\u003c/em\u003e Fairmaire (Coleoptera: Buprestidae), is a xylophagous buprestid, primary associated with trees of the genus \u003cem\u003eFraxinus\u003c/em\u003e. All European ash species are susceptible hosts of this beetle. This species is native to Asia (China, Japan, Korea, Mongolia and the Russian Far East) and has been accidentally introduced into North America and Western Russia, from where it has spread to Ukraine (Haak et., 2002, Liu et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2003\u003c/span\u003e, Baranchikov et., 2008, Drogvalenko et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, Evans et., 2020) and most recently to Belarus (Zviagintsev et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). \u003cem\u003eAgrilus planipennis\u003c/em\u003e is a quarantine pest listed in Part A of Annex II of Regulation (EU) 2019/2072. In addition, the European Commission carried out a risk assessment of these pests and identified 20 priority pests whose introduction can cause particularly high economic, ecological and social damage, including \u003cem\u003eA. planipennis\u003c/em\u003e (Regulation (EU) 2019/1702). A recent EFSA report on the potential impact of its introduction into the EU, estimated that 87.6% of ash trees could be lost, based on the considered scenario assumptions (EFSA, 2025).\u003c/p\u003e\u003cp\u003eHeavy infestations of \u003cem\u003eA. planipennis\u003c/em\u003e cause symptoms, such as smaller or discoloured leaves, progressive crown dieback, formation of epicormic shoots on branches and stems, bark cracking and flaking due to predation by woodpeckers and even the plant\u0026rsquo;s death. Furthermore, attacked trees show D-shaped insect exit holes of about 3\u0026ndash;4 mm, serpentine larval galleries under the bark, detached bark and debarking.\u003c/p\u003e\u003cp\u003eHowever, tree decay due to insect infestation can only be detected after years, when the population density has increased significantly. Indeed, \u003cem\u003eA. planipennis\u003c/em\u003e often remains undetected for several years in new environments (EFSA, 2024). These considerations suggest that surveillance, based on visual examination and sampling of plant parts, is not sufficient for the early detection of new EAB outbreaks. Recent experiences in North America indicate that trapping is the most effective method for EAB detection (EFSA, 2024). However, it must be considered that, in general, the morphological identification of \u003cem\u003eAgrilus\u003c/em\u003e species appears to be challenging (EPPO Global Database), especially due to the size of the genus, comprising more than 3000 species (Volkovitsh et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eMolecular identification of insects is a rising method, especially for quarantine species, for which rapid identification is crucial. In the case of xylophagous insects, the most frequently examined traces are frass samples in feeding tunnels or around entry/exit holes dug into trunks and branches (Rizzo et al., 2020, EPPO, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). DNA shed by organisms into environment samples is considered environmental DNA (eDNA). Molecular approaches based on eDNA offer several advantages: rapid detection via direct gene amplification, reduced reliance on taxonomic expertise, and minimal disturbance to the surveyed host. Numerous diagnostic protocols exploiting frass samples or other genetic residues have been developed in recent years (Ide et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, Kyei-Poku et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, Rizzo et al., 2020, Kyle et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eSpecific assays, targeting COI gene, to identify \u003cem\u003eA. planipennis\u003c/em\u003e and detect traces of its DNA within frass samples, were developed and validated on other European wood-borer species (Peterson et al. 2023, Kupper et al., 2025). In this study, we developed a new sensitive real-time qPCR assay, based on an alternative locus (Cytochrome B), with a Locked Nucleic Acid (LNA) probe for \u003cem\u003eA. planipennis\u003c/em\u003e detection. LNA probes and primers contain chemically modified bases that increase the rigidity of the ribose. This modification improves the stability, specificity, and affinity for base-pairing with target sequences (Josefsen et al., 2009). The high binding affinity allows for shorter probes, which results in higher binding specificity to the target DNA. The assay was validated using adult insect tissue and synthetic frass matrices, demonstrating its suitability for routine laboratory diagnostic applications.\u003c/p\u003e"},{"header":"2 Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003e2.1 Samples\u003c/h2\u003e\n \u003cp\u003eTarget and non-target organisms (including other genera present on the same host plants) were obtained from different sources and geographical origins (Table 1). The target specimens include 8 \u003cem\u003eA. planipennis\u003c/em\u003e adults and 6 artificial frass samples (obtained according to Rizzo et al., 2025). Among the 131 outgroup samples, 55 belong to \u003cem\u003eAgrilus\u003c/em\u003e spp. and 76 to other genera. To assess the efficiency and specificity of the assay a \u003cem\u003eGStrand\u003c/em\u003e (In 338) synthetic DNA (Eurofins Genomics), equivalent to the expected amplicon and based on the CytB gene of \u003cem\u003eA. planipennis\u003c/em\u003e (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e) was synthesised. All sample details are listed in Table 1.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003cdiv class=\"colspec\"\u003eTable 1. Samples from insects and frass used in this study.\u003c/div\u003e\n \u003ctable id=\"Taba\" border=\"1\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSpecies\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSample code\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eLife stage/sample type\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSupplier\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"15\"\u003e\n \u003cp\u003e\u003cem\u003eAgrilus planipennis*\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 000814\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"8\"\u003e\n \u003cp\u003eAdult\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"8\"\u003e\n \u003cp\u003eUSDA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 001563\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 001713\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 001638\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 001714\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 001714/2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 001714/3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 001714/4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR_Frass_Ap\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"6\"\u003e\n \u003cp\u003eArtificial Frass\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"6\"\u003e\n \u003cp\u003ePPS-T\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR_Frass_Ap/1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR_Frass_Ap/2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR_Frass_Ap/3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR_Frass_Ap/4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR_Frass_Ap/5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eIn 338\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003egStrand\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePPS-T\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cem\u003eAgrilus angustulus*\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 001710\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eAdult\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eJKI\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 001721/IIC\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"12\"\u003e\n \u003cp\u003e\u003cem\u003eAgrilus anxius*\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 001715\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"8\"\u003e\n \u003cp\u003eAdult\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"5\"\u003e\n \u003cp\u003eJKI\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 001716\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 001717\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 001718\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 001719\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 001720/(BBB11)\u003csup\u003e\u0026dagger;\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eRU\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 001721/(BBB12)\u003csup\u003e\u0026dagger;\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 001722/(BBB20)\u003csup\u003e\u0026dagger;\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGLFC\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 001723\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"4\"\u003e\n \u003cp\u003eArtificial Frass\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"4\"\u003e\n \u003cp\u003eJKI\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 001724\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 001725\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 001726\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"12\"\u003e\n \u003cp\u003e\u003cem\u003eAgrilus auroguttatus*\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 001701\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"8\"\u003e\n \u003cp\u003eAdult\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"8\"\u003e\n \u003cp\u003eUCa\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 001689\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 001689/2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 001689/3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 001689/4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 001689/5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 001689/6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 001689/7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 001689/F\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFrass\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eUCa\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR_Frass_Au\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eArtificial Frass\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003ePPS-T\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR_Frass_Au/1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 001689/Fc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFecal residue\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eUCa\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cem\u003eAgrilus biguttatus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 001722/IIC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eAdult\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eJKI\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 001524\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePPS-T\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cem\u003eAgrilus convexicollis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 001712\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eAdult\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eJKI\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 001720/IIC\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eAgrilus graminis*\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eIn 308\u003csup\u003e\u0026dagger;\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAdult\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eINRAE\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eAgrilus hastulifer\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eIn 309\u003csup\u003e\u0026dagger;\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAdult\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eINRAE\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cem\u003eAgrilus laticornis*\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 001709\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eAdult\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eJKI\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 001718\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eAgrilus obscuricollis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eIn 310\u003csup\u003e\u0026dagger;\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAdult\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eINRAE\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cem\u003eAgrilus olivicolor\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 001716\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eAdult\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eJKI\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 001717/1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eUoF\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eAgrilus sulcicollis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 001717\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAdult\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eJKI\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cem\u003eAgrilus viridis fagi\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 001711\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eAdult\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eUoF\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 001719\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eJKI\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"16\"\u003e\n \u003cp\u003e\u003cem\u003eAnoplophora chinensis*\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 001617\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"4\"\u003e\n \u003cp\u003eAdult\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"4\"\u003e\n \u003cp\u003ePPS-T\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 001618\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 001648\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 001620\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 000225\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLarva\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePPS-L\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 000259\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"5\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"5\"\u003e\n \u003cp\u003ePPS-T\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 001619\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 001678\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 001622\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 001649\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 000790\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"6\"\u003e\n \u003cp\u003eFrass\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"6\"\u003e\n \u003cp\u003ePPS-T\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 001621\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 001624\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 001639\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 001640\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 001668\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"4\"\u003e\n \u003cp\u003e\u003cem\u003eAnoplophora glabripennis*\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 000226\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAdult\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"4\"\u003e\n \u003cp\u003ePPS-L\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 000757\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLarva\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 000301\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eFrass\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 001625\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eAraecerus fasciculatus*\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 000824\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLarva\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePPS-T_PLI\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"8\"\u003e\n \u003cp\u003e\u003cem\u003eAromia bungii*\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 000260\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"4\"\u003e\n \u003cp\u003eAdult\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"5\"\u003e\n \u003cp\u003ePPS-C\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 000261\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 000262\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 001674\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 000254\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLarva\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 000231\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003eFrass\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePPS-C\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 000263\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eUoN\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 001688\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eAromia moschata\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 000766\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAdult\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eBuprestis cupressi\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 001707\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAdult\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eUoF\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cem\u003eCerambyx cerdo\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 000274\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eAdult\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eCREA_DC\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 000297\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cem\u003eCerambyx scopolii\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 000275\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eAdult\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eCREA_DC\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 000756\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eCerambyx welensii\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 000755\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAdult\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCREA_DC\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eCerambyx miles\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 000829\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAdult\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCREA_DC\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eChlorophorus glabromaculatus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 000799\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLarva\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePPS-T_PLI\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eChrysobothris affinis*\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 001944\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLarva\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePPS-T_PLI\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eChrysobothris igniventris*\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 001954\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLarva\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePPS-T_PLI\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cem\u003eChrysobothris quadriimpressa*\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 000853\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eLarva\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003ePPS-T_PLI\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 001694\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cem\u003eChrysobothris rugosiceps*\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 001699\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eLarva\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003ePPS-T_PLI\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 001700\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eChrysobothris solieri*\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 001702\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLarva\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePPS-T_PLI\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eChrysobotris femorata*\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 001615\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLarva\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePPS-T_PLI\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003e\u003cem\u003eCossus cossus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 000831\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAdult\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eUoP\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 000265\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLarva\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePPS-T\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 000289\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFrass\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePPS-T\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cem\u003eGraphisurus fasciatus*\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 000809\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eLarva\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003ePPS-T_PLI\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 001695\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eHylurgus ligniperda\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 000302\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAdult\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eUoF\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eHylurgopinus rufipes\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 000855\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLarva\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePPS-T_PLI\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eLarinus planus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 001923\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLarva\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePPS-T_PLI\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eLeptostylus asperatus*\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 001948\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLarva\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePPS-T_PLI\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eLepturges confluens*\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 001692\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLarva\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePPS-T_PLI\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003e\u003cem\u003eMonochamus galloprovincialis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 000250\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAdult\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCREA_DC\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 000258\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAdult\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePPS-L\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 000818\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAdult\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eUoF\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cem\u003eMonochamus sartor\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 000257\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAdult\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003ePPS-L\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 000256\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLarva\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eMorimus asper\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 000759\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAdult\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCREA_DC\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eNeoclytus acuminatus*\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 001704\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAdult\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eUoF\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eNeoclytus mucronatus*\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 000852\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLarva\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePPS-T_PLI\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cem\u003ePhryneta leprosa*\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 000828\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAdult\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003ePPS-T_PLI\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 000828/L\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLarva\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eSaperda carcharias\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 000767\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAdult\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eUoP\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eSaperda punctata*\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 000784\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAdult\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCREA_DC\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eSaperda scalaris*\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 000785\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAdult\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCREA_DC\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"4\"\u003e\n \u003cp\u003e\u003cem\u003eSaperda tridentata*\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 000294\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAdult\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"4\"\u003e\n \u003cp\u003ePPS-T_PLI\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 001690\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAdult\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 001691\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAdult\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 000247\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLarva\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eStictoleptura cordigera*\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 000792\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAdult\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eUoF\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cem\u003eZeuzera pyrina\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 000837\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLarva\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eUoP\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR 000838\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFrass\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003e\u003csup\u003e*\u003c/sup\u003eIdentified by sequence analysis of COI gene, using primers LCO1490/HCO2198 (Folmer et al., 1994).\u003c/p\u003e\n \u003cp\u003eProvided as DNA sample.\u003c/p\u003e\n \u003cp\u003e\u003csup\u003eb\u003c/sup\u003eUoF = University of Florence, Italy; JKI\u0026thinsp;=\u0026thinsp;Julius K\u0026uuml;hn Institute, Germany; UoP\u0026thinsp;=\u0026thinsp;University of Pisa, Italy; USDA\u0026thinsp;=\u0026thinsp;U.S. Department of Agriculture APHIS PPQ, Brighton, MI; UCa\u0026thinsp;=\u0026thinsp;University of California, USA; UoN\u0026thinsp;=\u0026thinsp;University of Naples, Italy; PPS-T\u0026thinsp;=\u0026thinsp;Plant Protection Service of Tuscany, Italy; PPS-T_PLI\u0026thinsp;=\u0026thinsp;Plant Protection Service of Tuscany, Port of Leghorn interception, Italy; CREA_DC\u0026thinsp;=\u0026thinsp;Consiglio per la Ricerca in agricoltura e l\u0026rsquo;analisi dell\u0026rsquo;Economia Agraria, Italy; PPS-C\u0026thinsp;=\u0026thinsp;Plant Protection Service_Campania, Italy; PPS-L\u0026thinsp;=\u0026thinsp;Plant Protection Service_Lombardy, Italy; RU\u0026thinsp;=\u0026thinsp;Rutgers University, USA; GLFC\u0026thinsp;=\u0026thinsp;Great Lakes Forestry Centre, Canada; INRAE\u0026thinsp;=\u0026thinsp;Laboratoire de Biologie des Ligneux et des Grandes Cultures, France.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003e2.2 DNA extraction\u003c/h2\u003e\n \u003cp\u003eDNA extraction was performed using a 2% CTAB buffer, followed by purification with Maxwell\u0026reg; RSC PureFood GMO and Authentication Kit (Promega Italia, Milan, Italy) (Rizzo et al., 2023). Artificial frass of EAB was produced by mixing CTAB lysates obtained from \u003cem\u003eFraxinus excelsior\u003c/em\u003e wood chips and from \u003cem\u003eA. planipennis\u003c/em\u003e adult specimen (sample MR001638 \u0026ndash; Table 1). Concentration and quality of the extracted DNA were quantified by using Qiaexpert (Qiagen). DNA obtained from both, target and non-target organisms (Table 1) was normalized to a concentration of 5 ng/\u0026micro;l and used for qPCR reactions and stored at -20\u0026deg;C until use. DNA amplifiability was verified by qPCR reactions using a TaqMan probe based on 18S rDNA region (Ioos et al., \u003cspan class=\"CitationRef\"\u003e2009\u003c/span\u003e) as described in Rizzo et al. (2025).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003e2.3 Primers and probe design for real-time PCR assay\u003c/h2\u003e\n \u003cp\u003eTo identify the specific genomic region suitable for oligonucleotide design, several sequences were analysed and evaluated. Their selection was based on the following factors: presence of polymorphisms, possibility of comparing similar sequences from other congeneric or genetically related species, use of specific mitochondrial genomic regions (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e) and highly conserved genes that provide interspecific variability (Kralik et al., 2017).\u003c/p\u003e\n \u003cp\u003eBased on the previous considerations and sequence availability, the CytB locus was selected.\u003c/p\u003e\n \u003cp\u003eCytB gene is characterized by low intraspecific variability (genetic distance\u0026thinsp;\u0026lt;\u0026thinsp;2%) and high interspecific divergence (distance\u0026thinsp;\u0026gt;\u0026thinsp;8%), allowing the distinction between cryptic or morphologically similar insect species (Chen et al., 2023). Primers and probe were designed, using the online software \u003cem\u003eOligoArchitec primer design\u003c/em\u003e (Sigma-Aldrich, St. Louis, USA), on the CytB region of \u003cem\u003eA. planipennis\u003c/em\u003e (KT363854.1) obtained from The National Center for Biotechnology Information (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov\u003c/span\u003e\u003c/span\u003e). The primers and LNA probe were synthesized by Eurofins Genomics (Ebersberg, Germany) (Fig. 2).\u003c/p\u003e\n \u003cp\u003eTo evaluate the \u003cem\u003ein silico\u003c/em\u003e specificity, a total of 640 sequences from 103 different \u003cem\u003eAgrilus\u003c/em\u003e species were aligned and compared with the newly designed amplicon (Figure \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e, supplementary material) by using the Geneious software. These sequences belong to species with the greatest genetic affinity, similar ecological niches or the potential for morphological misidentification (Volkovitsh et al., \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e). The amplicon specificity was verified by using the BLAST software (Basic Local Alignment Search Tool; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.ncbi.nlm.nih.gov/BLAST\u003c/span\u003e\u003c/span\u003e) and a partial sequences alignment is shown in Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003e2.4 Optimisation and validation of the qPCR method\u003c/h2\u003e\n \u003cp\u003eBased on the melting temperatures of the oligos, the real-time PCR probe protocol has been optimized for the annealing temperature by preparing a gradient (between 52\u0026deg;C and 62\u0026deg;C). The oligo concentration was evaluated as follows: 0.1 \u0026micro;M, 0.2 \u0026micro;M, 0.3 \u0026micro;M, and 0.4 \u0026micro;M for the primers and probe. For each run, two tubes containing nuclease-free distilled water (no template control - NTC), a positive and a negative amplification control were tested. Validation was performed according to the EPPO Standard PM7/98 (5). The specificity of the qPCR assay was tested for all samples listed in Table\u0026nbsp;1 by normalising their DNA concentration to 5 ng/\u0026micro;L.\u003c/p\u003e\n \u003cp\u003eThe limit of detection (LoD) was determined on 1:5 serial dilutions of target DNA extracts (\u003cem\u003eA. planipennis\u003c/em\u003e adult) ranging between 10 ng/\u0026micro;L to 5.12 fg/\u0026micro;L. Each dilution point was analysed in triplicate. The standard curve generated from the same serial dilution was used to quantify the target DNA in the artificial frass. Intra-variation (repeatability) and inter-variation (reproducibility) were performed and evaluated based on mean Cq values and their standard deviations (Dhami et al., \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e, Groth-Helms et al., 2023). Repeatability and reproducibility were performed using DNA extracts from \u003cem\u003eA. planipennis\u003c/em\u003e adult, normalized to a concentration of 16 pg/\u0026micro;L.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003e2.5 Blind test\u003c/h2\u003e\n \u003cp\u003eThe blind test was carried out with 12 blind samples, at different DNA concentrations (10, 2 and 0.5 ng/\u0026micro;L): 6x \u003cem\u003eA. planipennis\u003c/em\u003e, 2x \u003cem\u003eA. anxius\u003c/em\u003e, 2x \u003cem\u003eA. sulcicollis\u003c/em\u003e, 1x \u003cem\u003eA. graminis\u003c/em\u003e, 1x Negative Template control (NTC). Each sample was numbered progressively and processed in triplicates. The laboratories involved in the blind panel were: 1) LAB 1 (organizer): Phytopathological laboratory of the Tuscany Regional Phytosanitary Service, headquarters of Pistoia (Italy); 2) LAB 2: Phyto pathological laboratory of the Tuscany Regional Phytosanitary Service, headquarters of Livorno (Italy); 3) LAB 3: Phytopathological laboratory of the Tuscany Regional Phytosanitary Service, headquarters of Florence (Italy); LAB 4: laboratory of the CNR- IPSP of Sesto Fiorentino\u0026mdash;Florence (Italy).\u003c/p\u003e\n \u003cp\u003eEach laboratory conducted the qPCR analyses using the same diagnostic protocol developed in this study, but with different instruments and master mixes: i) LAB 1_ CFX96 (Bio-Rad) thermocycler and Quantinova Probe (Qiagen) master mix; ii) LAB 2: ARIA MMX (Agilent) thermocycler and Luna universal Probe (NEB) Master mix; iii) LAB. 3: Line Gene 96 Plus (Bioer, Rome, Italy) thermocycler and Quantinova Probe (Qiagen) master mix; iv) LAB 4: Rotor-Gene Q (Qiagen) thermocycler and TaqMan Universal PCR Master Mix (Applied Biosystems).\u003c/p\u003e\n \u003cp\u003eThe evaluation of the performance of the participating laboratories was assessed by considering the qualitative values (Positive/Negative) recorded in the PT results sheet (Chabirand et al., \u003cspan class=\"CitationRef\"\u003e2014\u003c/span\u003e). The results were interpreted for each laboratory by calculating the number of true positives (TP), true negatives (TN), false positives (FP) and false negatives (FN), according to the Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\n \u003ctable id=\"Tab1\" border=\"1\" class=\"fr-table-selection-hover\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eParameters considered in the blind panel: sensitivity, specificity and diagnostic accuracy.\u0026nbsp;\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003ePerformance Criteria\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eDefinition\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eEquation\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDiagnostic sensitivity\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eConcordance between the analysis result and the assigned value of the samples, for which a POSITIVE value was assigned\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTP/(TP\u0026thinsp;+\u0026thinsp;FN) x 100%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDiagnostic specificity\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eConcordance between the analysis result and the assigned value of the samples, for which a NEGATIVE value was assigned\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTN/(TN\u0026thinsp;+\u0026thinsp;FP) x 100%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAccuracy\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eConcordance between analysis result and assigned value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(TP\u0026thinsp;+\u0026thinsp;TN)/N x 100%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003eTP: True positives; TN: True negatives; FP: False positives; FN: False negatives; N: Total number of samples.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Assay conditions of the qPCR LNA Probe protocol\u003c/h2\u003e\u003cp\u003eReal-time amplification reactions with the LNA probe were optimised considering the reaction mixture, primer annealing temperatures and optimal primer concentrations. The optimal reaction mixture consisted of the following reagents, in a final volume of 20 \u0026micro;L: 10 \u0026micro;L of QuantiNova Supermix 2x (Qiagen, Hercules, USA), with primer concentrations at 0,4 \u0026micro;M, 0,2 \u0026micro;M for LNA probe and 2 \u0026micro;L of DNA template. As for the optimal thermal protocol, the conditions are as follows: initial denaturation at 95\u0026deg;C for 2 minutes, followed by 40 cycles of 95\u0026deg;C for 15 s and annealing at 60\u0026deg;C for 40 s.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e3.2 DNA extraction\u003c/h2\u003e\u003cp\u003eQuality and amplifiability of extracted DNAs are shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e3\u003c/span\u003e. The Cq values were obtained by real-time PCR amplifying the ribosomal 18S gene (Ioos et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). A total of eight adult specimens of \u003cem\u003eA. planipennis\u003c/em\u003e and six samples of artificial frass were evaluated.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003ePerformance of DNA extraction from adult \u003cem\u003eAgrilus planipennis\u003c/em\u003e and artificial frass, based on mean concentrations of extracted DNA (\u0026plusmn;\u0026thinsp;SD), absorbance ratio (A260/280) and mean Cq values of 18S (Ioos et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSample\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMean DNA Concentration (ng/\u0026micro;L)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAbsorbance (A260/280)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eMean Cq (18S)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAdult (n\u0026thinsp;=\u0026thinsp;8)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e73.53\u0026thinsp;\u0026plusmn;\u0026thinsp;15.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e1.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e18.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eArtificial frass (n\u0026thinsp;=\u0026thinsp;6)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e49.22\u0026thinsp;\u0026plusmn;\u0026thinsp;2.72\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e1.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e24.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e3.3 qPCR assay validation\u003c/h2\u003e\u003cp\u003eNCBI BLAST\u003csup\u003e\u0026reg;\u003c/sup\u003e results showed a high specificity of the qPCR amplicon with \u003cem\u003eA. planipennis\u003c/em\u003e sequences (Identity\u0026thinsp;=\u0026thinsp;100%; E-value\u0026thinsp;=\u0026thinsp;1e-75); no relevant homology was found with any closely related species. The qPCR assay developed in this study provided comprehensive and exclusive results for \u003cem\u003eA. planipennis\u003c/em\u003e with a clear non-specificity towards the non-target organisms. The Limit of Detection (LoD) from \u003cem\u003eA. planipennis\u003c/em\u003e DNA adult was 25.6 fg/\u0026micro;L (Cq mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u0026thinsp;=\u0026thinsp;36.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28) (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003e\u0026ndash; \u003cem\u003eAgrilus planipennis\u003c/em\u003e analytical sensitivity assays using 1:5 serial dilutions (from \u003cb\u003e10\u003c/b\u003e ng/\u0026micro;L to \u003cb\u003e5.12\u003c/b\u003e fg/\u0026micro;L) in triplicate (A, B, and C). The Cq values are the mean of the three threshold cycles of each dilution.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eConcentration values\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eA\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eB\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eC\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eAverage Cq\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eSD (\u0026plusmn;)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e10 ng/\u0026micro;L\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e18.27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e18.42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e18.38\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e18.36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.06\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2 ng/\u0026micro;L\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e20.72\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e20.73\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e20.75\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e20.73\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.01\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e0.4 ng/\u0026micro;L\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e23.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e23.15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e23.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e23.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.03\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e0.08 ng/\u0026micro;L\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e25.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e25.35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e25.49\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e25.41\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.06\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e0.0016 ng/\u0026micro;L\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e27.53\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e27.61\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e27.56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e27.57\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.03\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3.2 pg/\u0026micro;L\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e30.38\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e30.26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e30.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e30.23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.13\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e0.64 pg/\u0026micro;L\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e32.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e32.22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e32.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e32.18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.05\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e0.13 pg/\u0026micro;L\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e34.94\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e34.94\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e34.87\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e34.92\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.03\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e25.6 fg/\u0026micro;L\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e36.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e36.87\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e36.23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e36.48\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.28\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5.12 fg/\u0026micro;L\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003en/a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003en/a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003en/a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003en/a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003en/a\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThe standard curve generated from 1:5 serial dilution \u003cem\u003eA. planipennis\u003c/em\u003e DNA showed the following parameters: correlation coefficient (R\u003csup\u003e2\u003c/sup\u003e): 0.999; slope: -3.3; Y-intercept: 21.7. The qPCR assay showed a 100% of efficiency (E) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe qPCR performed on \u003cem\u003eA. planipennis\u003c/em\u003e adult DNA revealed a repeatability and reproducibility of 100% (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eRepeatability and reproducibility values indicating replicates and corresponding mean (Cq\u0026thinsp;\u0026plusmn;\u0026thinsp;SD values) from \u003cem\u003eA. planipennis\u003c/em\u003e adult DNA (normalized at 16 pg/\u0026micro;L concentration).\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e\u003cp\u003e\u003cem\u003eRepeatability\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e\u003cp\u003e\u003cem\u003eReproducibility\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eReplicates\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTest A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTest B\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eTest C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eTest A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eTest B\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eTest C\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e28.24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e27.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e27.91\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e28.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e27.85\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e28.04\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e28.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e27.75\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e28.27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e27.74\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e27.81\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e28.03\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e27.72\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e27.87\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e27.97\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e27.83\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e27.92\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e27.92\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e27.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e27.77\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e28.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e28.26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e28.75\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e28.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e28.52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e27.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e28.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e29.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e28.4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e28.57\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e28.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e27.87\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e27.94\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e28.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e28.15\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e28.51\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e28.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e28.45\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e28.95\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e28.77\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e28.63\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e28.72\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e29.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e27.76\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e28.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e28.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e28.66\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eCq Mean\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e28.19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e28.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e28.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e28.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e28.30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e28.24\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eS.D.\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.41\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.37\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.48\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.27\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eDNA extracts from artificial \u003cem\u003eA. planipennis\u003c/em\u003e frass were diluted 1:10 up to the eighth dilution. \u003cem\u003eAgrilus planipennis\u003c/em\u003e DNA was detected up to the fifth dilution (Cq average\u0026thinsp;=\u0026thinsp;33.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17), that corresponds to 0.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 pg/\u0026micro;L (this value was found by interpolating the data from a standard curve using CFX Maestro 1.3 software). This LoD corresponds to the minimum amount of \u003cem\u003eA. planipennis\u003c/em\u003e DNA detectable indirectly via frass.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e3.4 Blind panel test\u003c/h2\u003e\u003cp\u003eThe blind panel test performed in 4 different laboratories showed a diagnostic sensitivity, specificity and accuracy of 100%. No false positives or false negatives were obtained (Table\u0026nbsp;6).\u003c/p\u003e\u003cp\u003eTable\u0026nbsp;6. Results of blind panel test from 4 different laboratories by using the qPCR probe LNA assay to detect \u003cem\u003eA. planipennis.\u003c/em\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Tabc\" border=\"1\"\u003e\u003ccolgroup cols=\"8\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSample Code\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSpecies\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eDNA Concentration\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eExpected result\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eLAB 1\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eLAB 2\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eLAB 3\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eLAB4\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAp1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eAgrilus planipennis\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10 ng/\u0026micro;L\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003ePositive\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cem\u003e+\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAp 2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eAgrilus planipennis\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2 ng/\u0026micro;L\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003ePositive\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cem\u003e+\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAp 3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eAgrilus graminis\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10 ng/\u0026micro;L\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eNegative\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cem\u003e-\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAp 4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eAgrilus anxius\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10 ng/\u0026micro;L\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eNegative\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cem\u003e-\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAp 5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eAgrilus planipennis\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.5 ng/\u0026micro;L\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003ePositive\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cem\u003e+\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAp 6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eAgrilus anxius\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10 ng/\u0026micro;L\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eNegative\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cem\u003e-\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAp 7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eAgrilus sulcicollis\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10 ng/\u0026micro;L\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eNegative\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cem\u003e-\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAp 8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eAgrilus planipennis\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10 ng/\u0026micro;L\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003ePositive\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cem\u003e+\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAp 9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eAgrilus sulcicollis\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10 ng/\u0026micro;L\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eNegative\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cem\u003e-\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAp 10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eAgrilus planipennis\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2 ng/\u0026micro;L\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003ePositive\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cem\u003e+\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAp 11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eAgrilus planipennis\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5 ng/\u0026micro;L\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003ePositive\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cem\u003e+\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAp 12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNTC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eNegative\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cem\u003e-\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eLAB 1: CFX96 (Bio-Rad) thermocycler and Quantinova Probe (Qiagen) master mix; LAB 2: ARIA MMX (Agilent) thermocycler and Luna universal Probe (NEB) master mix; LAB. 3: Line Gene 96 Plus (Bioer, Rome, Italy) thermocycler and Quantinova Probe (Qiagen) master mix; LAB 4: Rotor-Gene Q (Qiagen) thermocycler and TaqMan Universal PCR Master Mix (Applied Biosystems).\u003c/p\u003e\u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eThe presence of \u003cem\u003eA. planipennis\u003c/em\u003e poses a serious threat to ash forests and ecosystems in which these plants play a key role. Prevention, quarantine measures, surveillance, and early detection are essential to reduce its impact and limit its spread. Challenges related to morphological identification and detection at low population densities require the development of rapid, sensitive molecular tools for detecting traces of target DNA. The mitochondrial DNA genes cytochrome B (CytB) and cytochrome C oxidase subunit I (COI) are both used as barcode markers for insects (Ray et al., 2024). To date, molecular methods for detecting EAB have been designed based on cytochrome C oxidase subunit I (COI) (Kyei-Puku et al., 2020, Kyle et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2024\u003c/span\u003e, Kupper et al., 2025). To reduce the risk associated with non-specific amplification in eDNA analysis and make the results more reliable, it may be helpful to use a combination of assays developed on different gene regions.\u003c/p\u003e\u003cp\u003eIn this study, we developed an alternative qPCR assay using LNA based on another region of mitochondrial DNA, cytochrome B (CytB). The CytB genomic region provides sufficient variability to allow unequivocal diagnosis of the target species; the use of an LNA probe yielded results comparable to those obtained for the detection and identification of \u003cem\u003eA. anxius\u003c/em\u003e (Rizzo et al., 2025). The high specificity of the \u003cem\u003ein silico\u003c/em\u003e analysis results was confirmed by \u003cem\u003ein vivo\u003c/em\u003e experiments on target and non-target species. The sensitivity of the proposed qPCR protocol (LoD of 25.6 fg/\u0026micro;L with an average Cq of 36.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28) is comparable to that obtained with the LAMP test (20 fg/\u0026micro;L) (Peterson et al., 2023) on adult insects. This comparison with LAMP highlights the qPCR assay as a valid complementary molecular diagnostic tool. Given their distinct methodologies, both approaches are crucial for diagnostic confirmation and data reliability.\u003c/p\u003e\u003cp\u003eSimilar to other protocols for the indirect diagnosis of pests of interest, the developed protocol was able to detect genetic traces of the target organism in the environment (Kiewnick et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, Rizzo et al., 2024). For diagnosis based on frass produced by \u003cem\u003eA. planipennis\u003c/em\u003e larvae, the detection limit was 0.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 pg/\u0026micro;L. These values ensure that the indirect approach is reliable in verifying the presence of \u003cem\u003eA. planipennis\u003c/em\u003e in a given area. The robustness of the method was validated through proficiency tests conducted in four independent laboratories.\u003c/p\u003e\u003cp\u003eIn the context of national surveys or containment and/or eradication measures, to prevent the establishment and spread of \u003cem\u003eAgrilus planipennis\u003c/em\u003e within the EU (Regulation (EU) 2024/434), rapid and reliable identification is needed.\u003c/p\u003e\u003cp\u003eFor these reasons this method could be valuable at cross-border entry points and in high-risk areas (e.g. nurseries, parks), particularly within the European Union, which continues to face a significant risk of EAB introduction and spread.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contributions:\u003c/h2\u003e\u003cp\u003eConceptualization of the research approach, detailed laboratory methodologies, and experimental designs for this study were developed and conducted by D. Rizzo (principal investigator) with the assistance of C. G. Zubieta, A. Marrucci, B. Palmigiano, M. Moriconi, C. Ranaldi, N. Luchi, A. Santini, F. Pecori, L. Bartolini, M. Bracalini, B. Hoppe, S. Feltgen, and T. Panzavolta. All sample preparations, data collection, and statistical analyses of data collected from DNA extracts were completed by D. Rizzo, C. G. Zubieta, M. Moriconi, B. Palmigiano, and A. Marrucci. Data curation and data mining, reference assembly, and manuscript formatting were done by D. Rizzo, A. Gionni, N. Luchi, F. Pecori, A. Santini, S. Feltgen, T. Panzavolta, M. Bracalini and B. Hoppe. Writing of the original draft was prepared by D. Rizzo, A. Gionni, F. Pecori, A. Santini, N. Luchi, T. Panzavolta, M. Bracalini, S. Feltgen and B. Hoppe. Revisions of manuscripts were completed by all authors.\u003c/p\u003e\u003ch2\u003eACKNOWLEDGEMENTS\u003c/h2\u003e\u003cp\u003eThe authors are grateful to Dr. Benjamin Slager, supervising entomologist at the USDA APHIS PPQ, for providing the target insect specimens needed to set up the method.\u003c/p\u003e\u003ch2\u003eConflicts of Interest\u003c/h2\u003e\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBaranchikov Y, Mozolevskaya E, Yurchenko G, Kenis M (2008) Occurrence of the emerald ash borer, Agrilus planipennis in Russia and its potential impact on European forestry. EPPO Bull 38:233\u0026ndash;238. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1365-2338.2008.01234.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1365-2338.2008.01234.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChabirand A, Anthoine G, Pierson O, Hostachy B (2014) The organization of proficiency testing in plant pathology according to the ISO/IEC 17043: example of the French national reference laboratory. 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Acta Biologica Sibirica 11:847\u0026ndash;861. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.5281/zenodo.16744135\u003c/span\u003e\u003cspan address=\"10.5281/zenodo.16744135\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-plant-diseases-and-protection","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jpdp","sideBox":"Learn more about [Journal of Plant Diseases and Protection](https://www.springer.com/journal/41348)","snPcode":"41348","submissionUrl":"https://www.editorialmanager.com/jpdp","title":"Journal of Plant Diseases and Protection","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"emerald ash borer, quarantine pest, early detection, molecular diagnostic tool, phytosanitary surveillance","lastPublishedDoi":"10.21203/rs.3.rs-7980969/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7980969/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003eAgrilus planipennis\u003c/em\u003e is a quarantine pest that is threatening native ash populations in both North America and Europe. Early detection of this pest, which can cause severe infestations, is essential, and molecular tests applied at different developmental stages and environmental traces can contribute substantially to improving control measures to prevent or contain \u003cem\u003eA. planipennis\u003c/em\u003e infestations. In this study, a specific \u003cem\u003eA. planipennis\u003c/em\u003e real-time qPCR assay was developed using a Locked Nucleic Acid (LNA) probe based on the CytB (Cytochrome B) gene.\u003c/p\u003e\u003cp\u003eThe detection limit of this method was 25.6 fg/\u0026micro;l for adult DNA extracts and 0.21 pg/\u0026micro;l for frass produced by \u003cem\u003eA. planipennis\u003c/em\u003e larvae. The new qPCR probe test, which targets a different locus, not only allows identification of the pest and provides an indirect diagnosis through environmental DNA analysis but can also be used for cross-validation of results between different tests.\u003c/p\u003e","manuscriptTitle":"Novel Agrilus planipennis early-detection tool designed on Cytochrome B gene","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-18 10:27:37","doi":"10.21203/rs.3.rs-7980969/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Minor revisions","date":"2026-03-03T01:04:06+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2026-01-21T09:36:10+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-07T11:43:54+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Journal of Plant Diseases and Protection","date":"2025-10-31T12:37:31+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-31T08:04:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Plant Diseases and Protection","date":"2025-10-29T10:32:14+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-plant-diseases-and-protection","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jpdp","sideBox":"Learn more about [Journal of Plant Diseases and Protection](https://www.springer.com/journal/41348)","snPcode":"41348","submissionUrl":"https://www.editorialmanager.com/jpdp","title":"Journal of Plant Diseases and Protection","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"e846ccb6-d3fc-4363-bd09-2d95466e0ef4","owner":[],"postedDate":"November 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-06T10:12:14+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-18 10:27:37","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7980969","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7980969","identity":"rs-7980969","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}