Effect of the cleaning and disinfection methods on the hygienic conditions of fermentation tanks of table olives (Olea europaea L.) Negrinha de Freixo cultivar.

This study aimed to evaluate and identify the microbial community attached to the surfaces of fermenter tanks used in table olive Negrinha de Freixo cultivar processing through molecular analysis and verify if the cleaning/disinfection was done correctly. Four fermentation tanks previously used in table olive processing were sampled at three different inside areas: upper, middle, and lower. Before sampling, four cleaning/disinfection methods were applied to the tanks, including (i) pressurised water; (ii) a disinfectant product used to clean bowls (Vasiloxe); (iii) 10% sodium hydroxide solution (caustic soda liquid); and (iv) a disinfectant product used by the wine industry (Hosbit). For each sample collected, mesophilic aerobic bacteria, yeast and moulds (YMC), lactic acid bacteria (LAB), as well as total coliforms (TC) and Pseudomonas aeruginosa were evaluated. The results showed significant differences between the different cleaning/disinfection methods applied. The fermenter sanitised with only pressurised water showed a greater abundance of microorganisms than the others. Mesophilic aerobic bacteria were the predominant population, with counts ranging between 2.63 and 5.56 log10 CFU/100 cm2, followed by the moulds (3.11-5.03 log10 CFU/100 cm2) and yeasts (2.42-5.12 log10 CFU/100 cm2). High diversity of microbial communities was observed between the different fermenter tanks. The most abundant species belonged to Aureobasidium, Bacillaceae, Cladosporium, and Rhodotorula genera. LAB, TC, and P. aeruginosa were not detected. This study hopes to improve hygienic conditions and increase the quality assurance and safety of the final product.

The Digital Twin Paradigm Applied to Soil Quality Assessment: A Systematic Literature Review.

This article presents the results regarding a systematic literature review procedure on digital twins applied to precision agriculture. In particular, research and development activities aimed at the use of digital twins, in the context of predictive control, with the purpose of improving soil quality. This study was carried out through an exhaustive search of scientific literature on five different databases. A total of 158 articles were extracted as a result of this search. After a first screening process, only 11 articles were considered to be aligned with the current topic. Subsequently, these articles were categorised to extract all relevant information, using the preferred reporting items for systematic reviews and meta-analyses methods. Based on the obtained results, there are two main conclusions to draw: First, when compared with industrial processes, there is only a very slight rising trend regarding the use of digital twins in agriculture. Second, within the time frame in which this work was carried out, it was not possible to find any published paper on the use of digital twins for soil quality improvement within a model predictive control context.

The influence of bioclimate on soil microbial communities of cork oak.

BACKGROUND: Soil microbiomes are important to maintain soil processes in forests and confer protection to plants against abiotic and biotic stresses. These microbiomes can be affected by environmental changes. In this work, soil microbial communities from different cork oak Portuguese forests under different edaphoclimatic conditions were described by using a metabarcoding strategy targeting ITS2 and 16S barcodes. RESULTS: A total of 11,974 fungal and 12,010 bacterial amplicon sequence variants (ASVs) were obtained, revealing rich and diverse microbial communities associated with different cork oak forests. Bioclimate was described as the major factor influencing variability in these communities (or bioclimates/cork oak forest for fungal community), followed by boron and granulometry. Also, pH explained variation of fungal communities, while C:N ratio contributed to bacterial variation. Fungal and bacterial biomarker genera for specific bioclimates were described. Their co-occurrence network revealed the existence of a complex and delicate balance among microbial communities. CONCLUSIONS: The findings revealed that bacterial communities are more likely to be affected by different edaphoclimatic conditions than fungal communities, also predicting a higher impact of climate change on bacterial communities. The integration of cork oak fungal and bacterial microbiota under different bioclimates could be further explored to provide information about useful interactions for increasing cork oak forest sustainability in a world subject to climate changes.

Bacteria could help ectomycorrhizae establishment under climate variations.

Rhizosphere microbiome is one of the main sources of plant protection against drought. Beneficial symbiotic microorganisms, such as ectomycorrhizal fungi (ECMF) and mycorrhiza helper bacteria (MHB), interact with each other for increasing or maintaining host plant fitness. This mutual support benefits all three partners and comprises a natural system for drought acclimation in plants. Cork oak (Quercus suber L.) tolerance to drought scenarios is widely known, but adaptation to climate changes has been a challenge for forest sustainability protection. In this work, ECMF and MHB communities from cork oak forests were cross-linked and correlated with climates. Cenococcum, Russula and Tuber were the most abundant ECMF capable of interacting with MHB (ECMF~MHB) genera in cork oak stands, while Bacillus, Burkholderia and Streptomyces were the most conspicuous MHB. Integrating all microbial data, two consortia Lactarius/Bacillaceae and Russula/Burkholderaceae have singled out but revealed a negative interaction with each other. Russula/Burkholderaceae might have an important role for cork oak forest sustainability in arid environments, which will be complemented by the lower drought adaptation of competitive Lactarius/Bacillaceae. These microbial consortia could play an essential role on cork oak forest resilience to upcoming climatic changes.

Epiphytic and Endophytic Bacteria on Olive Tree Phyllosphere: Exploring Tissue and Cultivar Effect.

Variation on bacterial communities living in the phyllosphere as epiphytes and endophytes has been attributed to plant host effects. However, there is contradictory or inconclusive evidence regarding the effect of plant genetics (below the species' level) and of plant tissue type on phyllosphere bacterial community assembly, in particular when epiphytes and endophytes are considered simultaneously. Here, both surface and internal bacterial communities of two olive (Olea europaea) cultivars were evaluated in twigs and leaves by molecular identification of cultivable isolates, with an attempt to answer these questions. Overall, Proteobacteria, Actinobacteria and Firmicutes were the dominant phyla, being epiphytes more diverse and abundant than endophytes. Host genotype (at cultivar level) had a structuring effect on the composition of bacterial communities and, in a similar way, for both epiphytes and endophytes. Plant organ (leaf vs. twig) control of the bacterial communities was less evident when compared with plant genotype and with a greater influence on epiphytic than on endophytic community structure. Each olive genotype/plant organ was apparently selective towards specific bacterial operational taxonomic units (OTUs), which may lead to specific feedbacks on fitness of plant genotypes. Bacterial recruitment was observed to happen mainly within epiphytes than in endophytes and in leaves as compared with twigs. Such host specificity suggested that the benefits derived from the plant-bacteria interaction should be considered at genetic levels below the species.

GuttaFlow® Bioseal Cytotoxicity Assessment: In Vitro Study.

The sealers used for root canal treatment should be biocompatible for the peri-radicular tissues, to evaluate the cytotoxic effects of GuttaFlow® bioseal sealer and to compare them with AH26® epoxy resin. Culture media were conditioned with the GuttaFlow® bioseal and AH26® pellets. MDPC-23 odontoblast cell cultures were treated with conditioned medium and serial dilutions. To evaluate the metabolic activity and cellular viability, the MTT and SRB assays were performed. To determine the production of reactive oxygen species, the DHE and DCF-DA probes were used. Cell cycle and cell-death types were assessed by cytometry, and to evaluate the mineralization capacity, the Alizarin Red S coloration was used. Statistical analysis was performed using analysis of variance (ANOVA) when normality was found and Kruskal-Wallis on the opposite case. For the comparison with normality values, the Student t-test was used. Cells exposed to the GuttaFlow® bioseal conditioned medium maintained high metabolic activities, except at higher concentrations. Likewise, viability was maintained, but a significant decrease was observed after exposure to the highest concentration (p < 0.001), associated with cell death by late apoptosis and necrosis. When cell cultures were exposed to AH26®, metabolic activity was highly compromised, resulting in cell death. An imbalance in the production of peroxides and superoxide anion was observed. GuttaFlow® bioseal showed higher biocompatibility than AH26®.

Evaluation of the efficacy of dentin hypersensitivity treatments-A systematic review and follow-up analysis.

OBJECTIVES: To compare the treatments used to treat dentin hypersensitivity (DH), based on its efficacy and effect duration. METHODS: Medline/PubMed, Cochrane Library, EMBASE and ClinicalTrials were searched for articles published between 1 January 2008 and 14 November 2018, in English, Portuguese or Spanish, reporting clinical trials, completed and with results. This systematic review protocol was registered in PROSPERO, number CRD42019121986. RESULTS: Seventy-four randomised clinical trials were included in the systematic review, reporting patients from 16 to 65 years old, with a clinical diagnosis of DH, that evaluate the efficacy of a desensitising product, compared to pre-treatment, used the evaporative method stimulation and the visual analogue scale. These studies evaluated 5366 patients and at least 9167 teeth. Seven follow-up periods were considered corresponding to an immediate, medium or long-time effect. Sixty-six studies were included in the quantitative synthesis. Glutaraldehyde with HEMA, glass ionomer cements and Laser present significant immediate (until 7 days) DH reduction. Medium-term (until 1 month) reduction was observed in stannous fluoride, glutaraldehyde with HEMA, hydroxyapatite, glass ionomer cements and Laser groups. Finally, long-term significant reduction was seen at potassium nitrate, arginine, glutaraldehyde with HEMA, hydroxyapatite, adhesive systems, glass ionomer cements and LASER. CONCLUSIONS: All active ingredients show efficacy in DH reduction in different follow-up times. Only in-office treatments are effective in immediate DH reduction, maintaining its efficacy over time. For long-time effects, at-home treatments can also be used. More standardised evaluation protocols should be implemented to increase the robustly of the results.

Endophytic and Epiphytic Phyllosphere Fungal Communities Are Shaped by Different Environmental Factors in a Mediterranean Ecosystem.

The diversity and factors influencing fungal assemblages in phyllosphere of Mediterranean tree species have been barely studied, especially when endophytic and epiphytic communities are simultaneously considered. In this work, the endophytic and epiphytic fungal communities from olive tree phyllosphere were studied. This tree species is natural from the Mediterranean region and adapted to grow under adverse climatic conditions. The main objectives were to determine whether there are differences between both fungal communities and to examine whether different abiotic (climate-related) and biotic (plant organs) factors play a pivotal role in structuring these communities. Both communities differed in size and composition, with epiphytic community being richer and more abundant, displaying also a dominance of melanized fungi. Season was the major driver of community composition, especially of epiphytes. Other drivers shaping epiphytes were wind speed and temperature, while plant organ, rainfall, and temperature were the major drivers for endophytic composition. In contrast, canopy orientation caused slight variations in community composition of fungi, but with distinct effects in spring and autumn seasons. In conclusion, epiphytic and endophytic communities are not driven by the same factors. Several sources of variation undergo complex interactions to form and maintain phyllosphere fungal community in Mediterranean climates. Climatic parameters have influence on these fungal communities, suggesting that they are likely to be affected by climate changes in a near future.

Ectomycorrhizal fungal diversity and community structure associated with cork oak in different landscapes.

Cork oak (Quercus suber L.) forests play an important ecological and economic role. Ectomycorrhizal fungi (ECMF) are key components for the sustainability and functioning of these ecosystems. The community structure and composition of ECMF associated with Q. suber in different landscapes of distinct Mediterranean bioclimate regions have not previously been compared. In this work, soil samples from cork oak forests residing in different bioclimates (arid, semi-arid, sub-humid, and humid) were collected and surveyed for ectomycorrhizal (ECM) root tips. A global analysis performed on 3565 ECM root tips revealed that the ECMF community is highly enriched in Russula, Tomentella, and Cenoccocum, which correspond to the ECMF genera that mainly contribute to community differences. The ECMF communities from the rainiest and the driest cork oak forests were distinct, with soils from the rainiest climates being more heterogeneous than those from the driest climates. The analyses of several abiotic factors on the ECMF communities revealed that bioclimate, precipitation, soil texture, and forest management strongly influenced ECMF structure. Shifts in ECMF with different hyphal exploration types were also detected among forests, with precipitation, forest system, and soil texture being the main drivers controlling their composition. Understanding the effects of environmental factors on the structuring of ECM communities could be the first step for promoting the sustainability of this threatened ecosystem.

Antimicrobial activity of endophytic fungi from olive tree leaves.

In this study, the antimicrobial potential of three fungal endophytes from leaves of Olea europaea L. was evaluated and the host plant extract effect in the antimicrobial activity was examined. The volatile compounds produced by endophytes were identified by GC/MS and further correlated with the antimicrobial activity. In potato dextrose agar, both Penicillium commune and Penicillium canescens were the most effective inhibiting Gram-positive and -negative bacteria (up to 2.7-fold compared to 30 µg/mL chloramphenicol), whereas Alternaria alternata was most effective inhibiting yeasts (up to 8.0-fold compared to 25 µg/mL fluconazole). The presence of aqueous leaf extract in culture medium showed to induce or repress the antimicrobial activity, depending on the endophytic species. In the next step, various organic extracts from both A. alternata mycelium and cultured broth were prepared; being ethyl acetate extracts displayed the widest spectrum of anti-microorganisms at a minimum inhibitory concentration ≤0.095 mg/mL. The volatile composition of the fungi that displayed the highest (A. alternata) and the lowest (P. canescens) antimicrobial activity against yeasts revealed the presence of six volatiles, being the most abundant components (3-methyl-1-butanol and phenylethyl alcohol) ascribed with antimicrobial potentialities. Overall the results highlighted for the first time the antimicrobial potential of endophytic fungi from O. europaea and the possibility to be exploited for their antimicrobial agents.

Impact of a natural soil salinity gradient on fungal endophytes in wild barley (Hordeum maritimum With.).

Occurrence and distribution pattern of fungal endophytes in different tissues of halophytic plants across saline depressions are poorly studied. We investigated the endophytic fungal communities inhabiting roots, stems and leaves of Hordeum maritimum collected in a soil salinity gradient, i.e. non-saline, slightly saline and saline, using a culture-dependent approach. A total of 20 taxa belonging to Ascomycota phylum were identified by ITS rRNA gene sequence. Pyronema domesticum and Alternaria spp. were the most frequently isolated. Roots host higher diversity and were more frequently colonized by endophytes than aboveground organs. Endophytic composition of all organs surveyed differed according to salinity gradient. Contrary to expectations, the colonization rate of roots increased with soil salinity, indicating that under salt stress the endophyte-plant association is promoted. All the isolates exhibited in vitro saline tolerance, especially those belonging to genera Xylaria, Chalastospora, Alternaria and Pyronema. Fungal tolerance to NaCl under in vitro conditions appears to be more dependent on the isolates than on the sites of their isolation, suggesting that under natural conditions other factors, beyond soil salinity, should be taken into account. These findings highlight the importance of fungal endophytes in the protection and/or adaptation of both interacting species (plant-fungus) to salt stress under natural conditions.

A breakthrough on Amanita phalloides poisoning: an effective antidotal effect by polymyxin B.

Amanita phalloides is responsible for more than 90 % of mushroom-related fatalities, and no effective antidote is available. α-Amanitin, the main toxin of A. phalloides, inhibits RNA polymerase II (RNAP II), causing hepatic and kidney failure. In silico studies included docking and molecular dynamics simulation coupled to molecular mechanics with generalized Born and surface area method energy decomposition on RNAP II. They were performed with a clinical drug that shares chemical similarities to α-amanitin, polymyxin B. The results show that polymyxin B potentially binds to RNAP II in the same interface of α-amanitin, preventing the toxin from binding to RNAP II. In vivo, the inhibition of the mRNA transcripts elicited by α-amanitin was efficiently reverted by polymyxin B in the kidneys. Moreover, polymyxin B significantly decreased the hepatic and renal α-amanitin-induced injury as seen by the histology and hepatic aminotransferases plasma data. In the survival assay, all animals exposed to α-amanitin died within 5 days, whereas 50 % survived up to 30 days when polymyxin B was administered 4, 8, and 12 h post-α-amanitin. Moreover, a single dose of polymyxin B administered concomitantly with α-amanitin was able to guarantee 100 % survival. Polymyxin B protects RNAP II from inactivation leading to an effective prevention of organ damage and increasing survival in α-amanitin-treated animals. The present use of clinically relevant concentrations of an already human-use-approved drug prompts the use of polymyxin B as an antidote for A. phalloides poisoning in humans.

YEAST dynamics during the natural fermentation process of table olives (Negrinha de Freixo cv.).

Yeast population and dynamics associated to spontaneous fermentation of green table olives Negrinha de Freixo cv. were evaluated. Olives and brine samples were taken at different fermentation times, and yeast were enumerated by standard plate count and identified by sequencing of the internal transcribed spacer (ITS) region of the nuclear ribosomal DNA (rDNA). Saccharomyces cerevisiae was the most frequent, followed by Candida tropicalis, Pichia membranifaciens and Candida boidini, representing together 94.8% of the total isolates. Galactomyces reessii was also identified for the first time in table olives. The highest species diversity was found between 44 and 54 days of fermentation, both in brine and olive pulp. Furthermore, high similarity was observed between brine and olive pulp microbiotas. In conclusion, these results give valuable information to table olive industrials in order to achieve more knowledge on the fermentation process of this important Protected Designation of Origin product.

Determination of amatoxins and phallotoxins in Amanita phalloides mushrooms from northeastern Portugal by HPLC-DAD-MS.

Amanita phalloides is a toxic mushroom responsible for the majority of deaths occurring after mushrooms ingestion, mainly due to amatoxins. In the present study the contents and distribution of the major amatoxins and phallotoxins in different tissues of A. phalloides from two different sites of Portugal were analyzed by liquid chromatography (LC) coupled to diode array (DAD) and mass spectrometry (MS) detection. The main toxins were separated by LC and its chemical structures confirmed by MS. α-Amanitin contents in caps, stipe and volva tissues were quantified by RP-HPLC. The results show that caps have the highest content of amatoxins, whereas the volva was richest in phallotoxins. Moreover variability in the toxins composition from different geographic sites was also observed. This study provides for the first time the content of toxins in A. phalloides from Portugal.

Diversity, Composition, and Specificity of the Philaenus spumarius Bacteriome.

Philaenus spumarius (Linnaeus, 1758) (Hemiptera, Aphrophoridae) was recently classified as a pest due to its ability to act as a vector of the phytopathogen Xylella fastidiosa. This insect has been reported to harbour several symbiotic bacteria that play essential roles in P. spumarius health and fitness. However, the factors driving bacterial assemblages remain largely unexplored. Here, the bacteriome associated with different organs (head, abdomen, and genitalia) of males and females of P. spumarius was characterized using culturally dependent and independent methods and compared in terms of diversity and composition. The bacteriome of P. spumarius is enriched in Proteobacteria, Bacteroidota, and Actinobacteria phyla, as well as in Candidatus Sulcia and Cutibacterium genera. The most frequent isolates were Curtobacterium, Pseudomonas, and Rhizobiaceae sp.1. Males display a more diverse bacterial community than females, but no differences in diversity were found in distinct organs. However, the organ shapes the bacteriome structure more than sex, with the Microbacteriaceae family revealing a high level of organ specificity and the Blattabacteriaceae family showing a high level of sex specificity. Several symbiotic bacterial genera were identified in P. spumarius for the first time, including Rhodococcus, Citrobacter, Halomonas, Streptomyces, and Providencia. Differences in the bacterial composition within P. spumarius organs and sexes suggest an adaptation of bacteria to particular insect tissues, potentially shaped by their significance in the life and overall fitness of P. spumarius. Although more research on the bacteria of P. spumarius interactions is needed, such knowledge could help to develop specific bacterial-based insect management strategies.

Effect of photostimulation through red LED light radiation on natural fermentation of table olives: An innovative case study with Negrinha the Freixo variety.

In this study, for the first time, red LED light radiation was applied to the fermentation process of table olives using the Negrinha de Freixo variety. Photostimulation using LED light emission (630 ± 10 nm) is proposed to shorten and speed up this stage and reduce time to market. Several physical-chemical characteristics and microorganisms (total microbial count of mesophilic aerobic, molds, yeasts, and lactic acid bacteria) and their sequence during fermentation were monitored. The fermentation occurred for 122 days, with two irradiation periods for red LED light. The nutritional composition and sensory analysis were performed at the end of the process. Fermentation under red LED light increased the viable yeast and lactic acid bacteria (LAB) cell counts and decreased the total phenolics in olives. Even though significant differences were observed in some color parameters, the hue values were of the same order of magnitude and similar for both samples. Furthermore, the red LED light did not play a relevant change in the texture profile, preventing the softening of the fruit pulp. Similarly, LED light did not modify the existing type of microflora but increased species abundance, resulting in desirable properties and activities. The species identified were yeasts - Candida boidinii, Pichia membranifaciens, and Saccharomyces cerevisiae, and bacteria - Lactobacillus plantarum and Leuconostoc mesenteroides, being the fermentative process dominated by S. cerevisiae and L. plantarum. At the end of fermentation (122 days), the irradiated olives showed less bitterness and acidity, higher hardness, and lower negative sensory attributes than non-irradiated. Thus, the results of this study indicate that red LED light application can be an innovative technology for table olives production.

Pest categorisation of Malacosoma parallela.

The EFSA Panel on Plant Health performed a pest categorisation of Malacosoma parallela (Staudinger) (Lepidoptera: Lasiocampidae) for the territory of the European Union, following commodity risk assessments of Berberis thunbergii, Malus domestica, Prunus persica and P. dulcis plants for planting from Türkiye, in which M. parallela came to attention as of possible concern. M. parallela is commonly known as the mountain ring silk moth and is a polyphagous leaf-eating pest in west-central Asia, primarily feeding on deciduous trees and shrubs, and known to cause serious damage to Malus, Prunus, and Quercus species. It is found at a range of altitudes from 130 m to 3000 m although most common above 1000 m. It is a univoltine species. Eggs are laid in masses on twigs and branches in the summer and larvae hatch the following spring to feed on buds and fresh leaves. Host plants can be completely defoliated. Plants for planting and cut branches provide pathways for entry, especially if infested with egg masses. Host availability and climate suitability suggest that parts of the EU would be suitable for establishment. Adults can fly and the pest could spread naturally within the EU although adults only live for a few days. Faster and more extensive spread is therefore more likely via egg masses moved on plants for planting. The introduction of M. parallela into the EU could lead to outbreaks causing damage to deciduous trees and shrubs in forests and orchards. Phytosanitary measures are available to inhibit the entry and spread of this species. M. parallela satisfies all the criteria that are within the remit of EFSA to assess for it to be regarded as a potential Union quarantine pest.

Commodity risk assessment of Ligustrum ovalifolium and Ligustrum vulgare plants from the UK.

The European Commission requested the EFSA Panel on Plant Health to prepare and deliver risk assessments for commodities listed in Commission Implementing Regulation (EU) 2018/2019 as 'High risk plants, plant products and other objects'. This Scientific Opinion covers plant health risks posed by plants of the evergreen Ligustrum ovalifolium and the semi-evergreen Ligustrum vulgare imported from the United Kingdom (UK) as: (a) bare root plants and (b) plants in pots, taking into account the available scientific information, including the technical information provided by the UK. The category (a) 'bare root plants' includes bundles of 1- to 3-year-old bare root whips or transplants and single 1- to 7-year-old bare root plants. The category (b) 'plants in pots' includes bundles of 1- to 2-year-old cell grown plants (only L. vulgare) and 1- to 5-year-old plants in pots. All pests associated with the commodities were evaluated against specific criteria for their relevance for this opinion. Two EU quarantine pests, Bemisia tabaci and Scirtothrips dorsalis, and one pest not regulated in the EU, Diaprepes abbreviatus, fulfilled all relevant criteria and were selected for further evaluation. For the selected pests, the risk mitigation measures proposed in the technical dossier from the UK were evaluated taking into account the possible limiting factors. For these pests, an expert judgement is given on the likelihood of pest freedom considering the risk mitigation measures acting on the pest, including uncertainties associated with the assessment. In the assessment of risk, the age of the plants was considered, reasoning that older trees are more likely to be infested mainly due to longer exposure time and larger size. The degree of pest freedom varies among the pests evaluated, with B. tabaci being the pest most frequently expected on the imported plants. The Expert Knowledge Elicitation indicated with 95% certainty that between 9915 and 10,000 per 10,000 bare root plants and plants in pots will be free from B. tabaci.

Pest categorisation of Garella musculana.

The European Commission requested the EFSA Panel on Plant Health to conduct a pest categorisation of Garella musculana (Erschov) (Lepidoptera: Nolidae), following a commodity risk assessment of Juglans regia plants for planting from Türkiye, in which G. musculana was identified as a pest of possible concern to the European Union (EU). Commonly known as the Asian walnut moth, this pest is native to Central Asia and develops on shoots, buds and fruits of Juglans species such as the English walnut, J. regia and the black walnut, J. nigra. Other reported host plants, such as Prunus dulcis and Populus spp., still require confirmation. The pest was first recorded in the EU (Bulgaria) in 2016 and was then reported in Romania in 2018 and Italy in 2021. This moth completes from one to four generations per year depending on environmental conditions (from valley to mountain forests and orchards up to an altitude of 2100 m). Eggs are laid in groups of 2-3 on young nuts or on buds of 1-year-old shoots. Neonate larvae usually enter the young nut through the peduncle. After fully exploiting one nut, the larva continues feeding in another one. Development takes 25-40 days. Larvae of the autumn generation do not enter the nuts, and so feed only in the pericarp. Larvae also often feed inside 1-year-old shoots or leaf axils. Larvae develop within the host but exit to pupate under loose bark or in deep cracks of bark. The pest overwinters at the larval or pupal stages. Plants for planting, cut branches and infested nuts provide pathways for entry. Climatic conditions and availability of host plants in southern and central EU MSs have allowed this species to establish and spread in Bulgaria, Romania and Italy. Adults can fly and the pest could spread naturally within the EU. Impact on Juglans spp. cultivated for fruit, timber and ornamental purposes is anticipated. Phytosanitary measures are available to reduce the likelihood of entry and further spread of G. musculana. This species meets the criteria that are within the remit of EFSA to assess for this species to be regarded as a potential Union quarantine pest.

Pest categorisation of Popillia quadriguttata.

The EFSA Panel on Plant Health performed a pest categorisation of Popillia quadriguttata (Coleoptera: Scarabaeidae), following a commodity risk assessment of bonsai Pinus parviflora grafted onto P. thunbergii from China, in which P. quadriguttata was identified as a pest of possible concern for the territory of the European Union. This is a univoltine polyphagous pest that occurs in eastern Asia from Vietnam northwards through eastern China and Taiwan, South Korea and into Far East Russia. Hosts include species of fruit trees within the genera Malus and Prunus, trees of forestry and environmental importance such as Quercus and Ulmus, shrubs such as Wisteria, soft fruit such as Rubus, grasses, including amenity turf and field crops such as potatoes, maize and soybean. Adults feed on host leaves, tender stems, flower buds, flowers and fruits; larvae feed on host roots. In northern China P. quadriguttata is a major pest of soybean; in South Korea, P. quadriguttata is one of the most serious insect pests of golf course turf. P. quadriguttata could enter the EU on various pathways including infested soil and growing media accompanying host plants for planning. Biotic factors (host availability) and abiotic factors (climate suitability) suggest that large parts of the EU would be suitable for establishment. Local spread would be mainly via natural dispersal of adults. Long distance spread would be facilitated by the movement of eggs, larvae and pupae infesting soil especially with plants for planting; adults could spread on plants for planting without soil. Economic and or environmental impacts would be expected on a range of plants if P. quadriguttata were to establish in the EU. Phytosanitary measures are available to reduce the likelihood of its introduction. P. quadriguttata satisfies all of the criteria that are within the remit of EFSA to assess for it to be regarded as a potential Union quarantine pest.