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Anoplophora glabripennis (Motschulsky 1853) (Coleoptera: Cerambycidae), the Asian Longhorned Beetle, is native to temperate and subtropical areas of China and the Korean peninsula. Due to its wide range of host plants, it is considered among the most economically important invasive plant pests. The morphological identification of A. glabripennis larvae can be confirmed by DNA barcoding, but obtaining the specimens from infested trees can be a demanding and challenging task. Therefore, non-invasive diagnostic tools based on DNA extracted from frass samples can be of key importance in phytosanitary surveys. In this study, an in silico generated real-time quantitative PCR test was developed for the detection of A. glabripennis DNA from frass material, which is naturally extruded from larval tunnels through cracks in the bark. Specificity was confirmed against a wide range of other wood-boring insect species frequently encountered during phytosanitary surveys and inclusivity was demonstrated for different populations of A. glabripennis from all main European outbreak areas. The test proved sensitive and reliable in detecting A. glabripennis DNA extracted from woody frass material of Acer saccharinum and Aesculus hippocastanum at least up to the 100-fold dilution. Furthermore, the test allowed the molecular identification of any life stage of the insect, including eggs and young larvae, whose morphological identification is impossible or very challenging. This study provides a reliable and sensitive molecular tool to detect A. glabripennis DNA in woody frass material, thus allowing a non-invasive sampling approach.

Magnetic resonance imaging (MRI) often requires contrast agents to improve the visualization in some tissues and organs, including the gastrointestinal tract. In this latter case, instead of intravascular administration, oral agents can be used. Natural oral contrast agents, such as fruit juice, have the advantages of better taste, tolerability, and lower price with respect to the artificial agents. We have characterized the relaxometry profiles of pineapple juice in order to understand the origin of the increase in relaxation rates (and thus of the MRI contrast) in reference to its content of manganese ions. Furthermore, we have characterized the relaxometry profiles of pineapple juice in the presence of alginate in different amounts; the interaction of the manganese ions with alginate slows down their reorientation time to some extent, with a subsequent increase in the relaxation rates. The relaxometry profiles were also compared with those of manganese(II) solutions in 50 mmol/dm3 sodium acetate solution (same pH of pineapple juice), which revealed sizable differences, mostly in the number of water molecules coordinated to the metal ion, their lifetimes, and in the constant of the Fermi-contact interaction. Finally, the fit of the transverse relaxivity shows that the increased viscosity in the hydrogel formulations can improve significantly the negative contrast of pineapple juice at the magnetic fields relevant for clinical MRI.

Recent structural studies show distinct morphologies for the fibrils of Aβ(1-42) and Aβ(1-40), which are believed not to co-fibrillize. We describe here a novel, structurally-uniform 1 : 1 mixed fibrillar species, which differs from both pure fibrils. It forms preferentially even when Aβ(1-42) : Aβ(1-40) peptides are mixed in a non-stoichiometric ratio.

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.

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.

Three molecular protocols using qPCR TaqMan probe, SYBR Green, and loop-mediated isothermal amplification (LAMP) methods were set up for the identification of larvae and adults of an African invasive moth, Thaumatotibia leucotreta (Meyrick, 1913) (Lepidoptera: Tortricidae). The DNA extracts from larval and adult samples of T. leucotreta were perfectly amplified with an average Ct value of 19.47 ± 2.63. All assays were demonstrated to be inclusive for T. leucotreta and exclusive for the nontarget species tested; the absence of false positives for nontarget species showed a 100% of diagnostic specificity and diagnostic sensitivity for all assays. With the SYBR Green protocol, the Cq values were only considered for values less than 22 (cutoff value) to prevent false-positive results caused by the late amplification of nonspecific amplicons. The limit of detection (LoD) for the qPCR probe protocol was equal to 0.02 pg/µl while a value equal to 0.128 pg/µl for the qPCR SYBR Green assay and LAMP method were established, respectively. The intrarun variabilities of reproducibility and repeatability in all the assays evaluated as CV%, ranged between 0.21 and 6.14, and between 0.33 and 9.52, respectively; the LAMP values were slightly higher than other assays, indicating a very low interrun variability. In order for an operator to choose the most desirable method, several parameters were considered and discussed. For future development of these assays, it is possible to hypothesize the setup of a diagnostic kit including all the three methods combined, to empower the test reliability and robustness.

Nuts of the sweet chestnut (Castanea sativa) are a widely appreciated traditional food in Europe. In recent years producers and consumers reported a drop of nut quality due to the presence of rot diseases caused by Gnomoniopsis smithogilvyi. Early detection of this pathogen is fundamental to the economic viability of the chestnut industry. In the present study, we developed three molecular methods based on real-time portable LAMP, visual LAMP and qPCR assays for G. smithogilvyi. The molecular assays were specific for G. smithogilvyi and did not amplify the other 11 Gnomoniopsis species and 11 other fungal species commonly associated with chestnuts. The detection limit of both the qPCR and real-time portable LAMP (P-LAMP) assays was 0.128 pg/µL, while the visual LAMP (V-LAMP) assay enabled the detection up to 0.64 pg/µL. By using these newly developed molecular tools, the pathogen was detected in symptomatic and asymptomatic nuts, but not in leaves. The reliability of these molecular methods, including the P-LAMP assay, was particularly useful in detecting G. smithogilvyi of harvested nuts in field, even in the absence of rot symptoms.

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.

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.

Biocompatible hydrogels for tissue regeneration/replacement and drug release with specific architectures can be obtained by three-dimensional bioprinting techniques. The preservation of the higher order structure of the proteins embedded in the hydrogels as drugs or modulators is critical for their biological activity. Solution nuclear magnetic resonance (NMR) experiments are currently used to investigate the higher order structure of biotherapeutics in comparability, similarity, and stability studies. However, the size of pores in the gel, protein–matrix interactions, and the size of the embedded proteins often prevent the use of this methodology. The recent advancements of solid-state NMR allow for the comparison of the higher order structure of the matrix-embedded and free isotopically enriched proteins, allowing for the evaluation of the functionality of the material in several steps of hydrogel development. Moreover, the structural information at atomic detail on the matrix–protein interactions paves the way for a structure-based design of these biomaterials.

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

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.

Aromia bungii (Faldermann, 1835) (Coleoptera: Cerambycidae), the red-necked longhorn beetle is native to eastern Asia, where it is an important wood-borer of fruit and ornamental species of the genus Prunus. A. bungii is a quarantine pest in the European Union, following its accidental introduction and establishment in Germany and Italy, and is currently included in the list of priority pests. To confirm its infestations in outbreak areas, adult or larval specimens are needed to perform morphological or molecular analyses. The presence of A. bungii larvae inside the attacked trees makes the collection of specimens particularly difficult. Thus, we present two diagnostic protocols based on frass analysis with real-time PCR (probe and SYBR Green). The results obtained show that a non-invasive approach for detecting the presence of this harmful invasive pest can be a reliable and accurate alternative diagnostic tool in phytosanitary surveys, as well as to outline a sustainable pest management strategy.

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.

The vector-borne bacterial pathogenXylella fastidiosais widely distributed in the Americas; in the last decade it has emerged as a serious threat for agricultural crops, natural environment and landscape in Europe. Following the first EU outbreak in 2013 in southern Italy, associated with a severe disease in olive trees, annual mandatory surveys are now in place in the Member States, leading to the discovery of bacterial outbreaks in different countries. Among the latest findings, an outbreak has been reported in the Italian region of Tuscany, with infections identified in seven different plant species. In this work, we report the isolation and the genetic characterization of isolates associated with this newly discovered outbreak. Multilocus sequence typing approach revealed the occurrence of isolates harbouring a new sequence type, denoted ST87, genetically related to strains of subsp.multiplex, but different from the genotypes of this subspecies previously characterized in Europe. Five cultured strains were successfully recovered from four of the seven host plants, an important achievement for advancing the studies on genomics and pathogenicity of theseisolates and thus assess their potential threat for European agriculture.

Due to its complex epidemiological cycle, including several polyphagous insect vectors and host plants, and the absence of efficient control strategies, Bois Noir (BN) disease of grapevine is encroaching wider territories in the main viticultural areas worldwide. Molecular approaches allowed to increase the knowledge about its etiological agent (Bois Noir phytoplasma, BNp; ‘Candidatus Phytoplasma solani’ species), revealing interesting features concerning BNp population structure and dynamics and transmission routes in vineyard agro‐ecosystems. In the present study, a multilocus sequence typing approach (vmp1 and stamp genes) was utilised for describing the genetic diversity among BNp strain populations in 17 vineyards localised in two distinct geographic areas in Tuscany (central Italy). The results confirmed that BNp ecology in Tuscan vineyards is mainly associated to the bindweed‐related host system, and allowed the identification of 14 BNp vmp1/stamp genotypes. Interestingly, the prevalent genotype (Vm43/St10) was never found in grapevines outside of Tuscany. Moreover, statistical analyses showed significant differences between the composition of BNp strain populations identified in grapevines from north‐western and central‐eastern Tuscany. These results reinforce the hypothesis that distinct geographic areas, probably associated with different ecological niches, can drive the selection of BNp strains, also favouring the entrance of unusual ‘Ca. Phytoplasma solani’ genotypes in vineyards.

Bois noir (BN) is the most widespread disease of the grapevine yellows complex in the Euro-Mediterranean area. BN is caused by ‘Candidatus Phytoplasma solani’ (BNp), transmitted from herbaceous plants to grapevine by polyphagous insect vectors. In this study, genetic diversity among BNp strains and their prevalence and possible association with grapevine symptom severity were investigated in a Sangiovese clone organic vineyard in the Chianti Classico area (Tuscany). Field surveys over 2 years revealed a range of symptom severity on grapevine and an increase of BN incidence. A TaqMan allelic discrimination assay detected only tufB type b among BNp strains, suggesting the prevalence of the bindweed-related ecology. Nucleotide sequence analyses of vmp1 and stamp genes identified 12 vmp1 and 16 stamp sequence variants, showing an overall positive selection for such genes. The prevalent genotype was Vm43/St10, reported for the first time in this study and closely related to strains identified only in the French Eastern Pyrenees. BNp strains identified in the examined vineyard and mostly grouped in separate bindweed-related phylogenetic clusters showed statistically significant differences in their distribution in grapevines exhibiting distinct symptom severity. These results suggest the possible occurrence of a range of virulence within BNp strain populations in the Chianti Classico area.

Citrus tristeza closterovirus (CTV) has been found several times in the last decades in Italy, and plant protection services are involved in monitoring and surveillance. Although orchards linked to the citrus industry are well monitored, there is an underestimated risk of viruses or virus-like diseases in ornamental nurseries. Our aim was to modify a CTV monitoring program to include other viruses (Citrus variegation virus, CVV; Citrus psorosis virus, CPsV) and viroids (Citrus exocortis viroid, CEVd; Hop stunt viroid, HSVd; Citrus bent leaf viroid, CBLVd; Citrus dwarfing viroid, CDVd; Citrus bark cracking viroid, CBCVd). Ornamental mother plants were monitored for four years in 15 nurseries in two locations in central Italy using inexpensive multiplex RT-PCR protocols. CTV incidence was 1.6–13.5%, with an average distribution of 11.9%. The average incidence of CVV and CPsV was 6.3% and 2.7%, respectively. Higher CTV, CVV and CPsV incidences were observed in C. x paradisi, C. grandis and C. x clementina. The most widespread viroid identified was CEVd (32.9%), frequently observed in C. x limonia and C. limon. HSVd (10.5%), and CDVd (7.1%) were mostly found in C. x limonia. Lower infection rates were observed for CBLVd (2.0%) and CBCVd (1.4%). However, the nurseries’ response to the virus alert by the protection services was only partially effective. Although the CTV incidence was lower in nurseries re-checked after the initial detection, it was not eradicated from two nurseries out of three, and the occurrence of viroids was reduced in just one nursery. Given that dangerous viruses along with the concomitant spread of viroids have unfortunately become a fact of every day life, multiplex RT-PCR diagnoses are likely to play an increasing role in warning nursery managers of possible infections.