ABSTRACT
The use of molecular tools to identify insect pests is a critical issue, especially when rapid and reliable tests are required. We proposed a protocol based on qPCR with SYBR Green technology to identify Philaenus italosignus (Hemiptera, Aphrophoridae). The species is one of the three spittlebugs able to transmit Xylella fastidiosa subsp. pauca ST53 in Italy, together with Philaenus spumarius and Neophilaenus campestris. Although less common than the other two species, its identification is key to verifying which role it can play when locally abundant. The proposed assay shows analytical specificity being inclusive with different populations of the target species and exclusive with non-target taxa, either taxonomically related or not. Moreover, it shows analytical sensibility, repeatability, and reproducibility, resulting in an excellent candidate for an official diagnostic method. The molecular test can discriminate P. italosignus from all non-target species, including the congeneric P. spumarius.
ABSTRACT
DNA extraction from plant samples is very important for a good performance of diagnostic molecular assays in phytopathology. The variety of matrices (such as leaves, roots, and twigs) requires a differentiated approach to DNA extraction. Here we describe three categories of matrices: (a) symptomatic bark/wood tissue; (b) residues of frass resulting from insect woody trophic activities, portions of the galleries produced in the wood, and tissues surrounding exit holes; and (c) leaves of different plant species. To improve the performances of diagnostic assays, we here describe DNA extraction procedures that have been optimized for each matrix type.
Subject(s)
Plant Leaves , Plants , DNA, Plant/analysis , DNA, Plant/genetics , Plant Leaves/chemistry , Plant Leaves/genetics , Plant Roots/genetics , Plants/genetics , WoodABSTRACT
Walnut species (Juglans spp.) are multipurpose trees, widely employed in plantation forestry for high-quality timber and nut production, as well as in urban greening as ornamental plants. These species are currently threatened by the thousand cankers disease (TCD) complex, an insect-fungus association which involves the ascomycete Geosmithia morbida (GM) and its vector, the bark beetle Pityophthorus juglandis. While TCD has been studied extensively where it originated in North America, little research has been carried out in Europe, where it was more recently introduced. A key step in research to cope with this new phytosanitary emergency is the development of effective molecular detection tools. In this work, we report two accurate molecular methods for the diagnosis of GM, based on LAMP (real-time and visual) and SYBR Green qPCR, which are complimentary to and integrated with similar recently developed assays. Our protocols detected GM DNA from pure mycelium and from infected woody tissue with high accuracy, sensitivity, and specificity, without cross-reactivity to a large panel of taxonomically related species. The precision and robustness of our tests guarantee high diagnostic standards and could be used to support field diagnostic end-users in TCD monitoring and surveillance campaigns.
ABSTRACT
A molecular diagnostic method using TaqMan probe qPCR is presented for the identification of Anoplophora chinensis (Förster) (Coleoptera: Cerambycidae) from whole body insects (adults and larvae) and frass samples stored under different conditions. The results showed a perfect amplification of DNA from all samples; the repeatability and reproducibility of the protocol were very good, with standard deviations of inter-run and intra-run variability less than or equal to 0.5. The assay allowed to discern all A. chinensis samples from those of the other non-target wood-borer species, with 100% correspondence to the homologous sequences. No amplification or cross reactions were observed with A. glabripennis (Motschulsky) (Coleoptera: Cerambycidae), which is the most related species among those tested. The protocol was validated by an internal blind panel test which showed a good correspondence between the results obtained by different operators in the same lab. The analytical sensitivity for the lab frass with the Probe qPCR, namely the lowest amount of A. chinensis DNA that can be detected (LoD), was 0.64 pg/µl with a Cq of 34.87. The use of indirect evidence for the identification of a pest is an important feature of the method, which could be crucial to detect the presence of wood-boring insects. This diagnostic tool can help prevent the introduction of A. chinensis into new environments or delimit existing outbreak areas thanks to indirect frass diagnosis.
Subject(s)
Coleoptera , Animals , Coleoptera/genetics , Larva , Reproducibility of Results , WoodABSTRACT
Molecular assays based on qPCR TaqMan Probes were developed to identify three species of the genus Xylosandrus, X. compactus, X. crassiusculus and X. germanus (Coleoptera Curculionidae Scolytinae). These ambrosia beetles are xylophagous species alien to Europe, causing damages to many ornamental and fruiting trees as well as shrubs. DNA extraction was carried out from adults, larvae and biological samples derived from insect damages on infested plants. For X. compactus, segments of galleries in thin infested twigs were cut and processed; in the case of X. crassiusculus, raw frass extruded from exit holes was used, while DNA of X. germanus was extracted from small wood chips removed around insect exit holes. The assays were inclusive for the target species and exclusive for all the non-target species tested. The LoD was 3.2 pg/µL for the frass of X. crassiusculus and 0.016 ng/µL for the woody matrices of the other two species. Both repeatability and reproducibility were estimated on adults and woody samples, showing very low values ranging between 0.00 and 4.11. Thus, the proposed diagnostic assays resulted to be very efficient also on the woody matrices used for DNA extraction, demonstrating the applicability of the protocol in the absence of dead specimens or living stages.
ABSTRACT
Tomato brown rugose fruit virus (ToBRFV) represents an emerging viral threat to the productivity of tomato and pepper protected cultivation worldwide. This virus has got the status of quarantine organism in the European Union (EU) countries. In particular, tomato and pepper seeds will need to be free of ToBRFV before entering the EU and before coming on the market. Thus, lab tests are needed. Here, we develop and validate a one-step reverse transcription LAMP platform for the detection of ToBRFV in tomato and pepper leaves, by real-time assay [reverse transcription loop-mediated isothermal amplification (RT-LAMP)] and visual screening (visual RT-LAMP). Moreover, these methods can also be applied successfully for ToBRFV detection in tomato and pepper seeds. The diagnostic specificity and sensitivity of both RT-LAMP and visual RT-LAMP are both 100%, with a detection limit of nearly 2.25 fg/µl, showing the same sensitivity as RT-qPCR Sybr Green, but 100 times more sensitive than end-point RT-PCR diagnostic methods. In artificially contaminated seeds, the proposed LAMP assays detected ToBRFV in 100% of contaminated seed lots, for up to 0.025-0.033% contamination rates in tomato and pepper, respectively. Our results demonstrate that the proposed LAMP assays are simple, inexpensive, and sensitive enough for the detection of ToBRFV, especially in seed health testing. Hence, these methods have great potential application in the routine detection of ToBRFV, both in seeds and plants, reducing the risk of epidemics.
ABSTRACT
The red-necked longhorn beetle Aromia bungii (Faldermann, 1835) (Coleoptera: Cerambycidae) is native to east Asia, where it is a major pest of cultivated and ornamental species of the genus Prunus. Morphological or molecular discrimination of adults or larval specimens is required to identify this invasive wood borer. However, recovering larval stages of the pest from trunks and branches causes extensive damage to plants and is timewasting. An alternative approach consists in applying non-invasive molecular diagnostic tools to biological traces (i.e., fecal pellets, frass). In this way, infestations in host plants can be detected without destructive methods. This paper presents a protocol based on both real-time and visual loop-mediated isothermal amplification (LAMP), using DNA of A. bungii extracted from fecal particles in larval frass. Laboratory validations demonstrated the robustness of the protocols adopted and their reliability was confirmed performing an inter-lab blind panel. The LAMP assay and the qPCR SYBR Green method using the F3/B3 LAMP external primers were equally sensitive, and both were more sensitive than the conventional PCR (sensitivity > 103 to the same starting matrix). The visual LAMP protocol, due to the relatively easy performance of the method, could be a useful tool to apply in rapid monitoring of A. bungii and in the management of its outbreaks.
ABSTRACT
The cultivation of walnuts (Juglans sp.) in Europe retains high economic, social, and environmental value. The recent reporting of the Thousand Cankers Disease (TCD) fungus, Geosmithia morbida, and of its vector, Pityophthorus juglandis, in walnut trees in Italy is alarming the whole of Europe. Although Italy is at present the only foothold of the disease outside North America, given the difficulties inherent in traditional identification of both members of this beetle/fungus complex, a rapid and effective protocol for the early detection and identification of TCD organisms is an absolute priority for Europe. Here we report the development of an effective and sensitive molecular tool based on simplex/duplex qPCR assays for the rapid, accurate and highly specific detection of both the bionectriaceous fungal pathogen and its bark-beetle vector. Our assay performed excellently, detecting minute amounts of target DNA without any non-specific amplification. Detection limits from various and heterogeneous matrices were lower than other reported assays. Our molecular protocol could assist in TCD organism interception at entry points, territory monitoring for the early identification and eradication of outbreaks, delineation of quarantine areas, and tracing back TCD entry and dispersal pathways.
Subject(s)
DNA, Environmental/isolation & purification , Hypocreales/genetics , Juglans/microbiology , Real-Time Polymerase Chain Reaction , Weevils/genetics , Animals , DNA, Environmental/genetics , DNA, Fungal/isolation & purification , Insect Vectors/genetics , Insect Vectors/microbiology , Italy , Limit of Detection , Plant Diseases/microbiology , Plant Diseases/prevention & control , Reproducibility of Results , Weevils/microbiologyABSTRACT
Anoplophora glabripennis (Motschulsky, 1853), native to eastern Asia, is a destructive woodborer of many ornamental species, leading to the decline and the death of the attacked trees. In outbreak areas as Europe or North America, this pest is usually identified using morphological or molecular analyses of adult or larval specimens. However, the procedures for collecting A. glabripennis specimens from infested plants are too expensive and time consuming for routine screening. A noninvasive diagnostic tool based on frass discrimination is therefore crucial for the rapid identification of A. glabripennis at different development stages in the host. This article describes a rapid diagnostic protocol based on loop-mediated isothermal amplification (LAMP). DNA extracted from A. glabripennis frass was amplified with both visual and real-time LAMP and compared with those of nontarget species. The results show that the method is reliable and accurate and therefore could be a promising diagnostic tool in phytosanitary surveys.