Aggressiveness and Patulin Production in Penicillium expansum Multidrug Resistant Strains with Different Expression Levels of MFS and ABC Transporters, in the Presence or Absence of Fludioxonil.

Penicillium expansum is the most common postharvest pathogen of apple fruit, causing blue mold disease. Due to the extensive use of fungicides, strains resistant to multiple chemical classes have been selected. A previous study by our group proposed that the overexpression of MFS (major facilitator superfamily) and ABC (ATP binding cassette) transporters constitute an alternative resistance mechanism in Multi Drug resistant (MDR) strains of this pathogen. This study was initiated to determine two main biological fitness parameters of MDR strains: aggressiveness against apple fruit and patulin production. In addition, the expression pattern of efflux transporters and hydroxylase-encoding genes that belong to the patulin biosynthesis pathway, in the presence or absence of fludioxonil and under in vitro and in vivo conditions were investigated. Results showed that the MDR strains produced higher concentrations of patulin but showed a lower pathogenicity compared to the wild-type isolates. Moreover, expression analysis of patC, patM and patH genes indicated that the higher expression levels do not correlate with the detected patulin concentration. The selection of MDR strains in P. expansum populations and the fact that they produce more patulin, constitutes a serious concern not only for successful disease control but also for human health. The above-mentioned data represent the first report of MDR in P. expansum associated with its patulin-production ability and the expression level of patulin biosynthesis pathway genes.

An Optimized HPLC-DAD Methodology for the Determination of Anthocyanins in Grape Skins of Red Greek Winegrape Cultivars (Vitis vinifera L.).

A rapid and simple HPLC-DAD analytical method was developed and optimized for the determination of anthocynanins in three red Greek winegrape varieties (Kotsifali, Limnio, and Vradiano). The critical parameters, such as the acidifying solvent and the extraction temperature, which affect the extraction of anthocyanins from the grapes, were studied to find the optimum values. The developed methodology was validated in terms of selectivity, linearity, accuracy, and precision and presented satisfactory results. The limits of quantification (LOQs) ranged between 0.20 mg/kg to 0.60 mg/kg, and the limits of detection (LODs) ranged between 0.06 mg/kg and 0.12 mg/kg. The RSD% of the within-day and between-day assays were lower than 6.2% and 8.5%, respectively, showing adequate precision. The accuracy ranged between 91.6 and 119% for within-day assay and between 89.9 and 123% for between-day assay. Sixteen samples from the main regions of each variety as well as from the official ampelographic collections of Greece were collected during the 2020 growing season and were further analyzed by HPLC-DAD. Notable differences in the anthocyanin content were detected among the cultivars using hierarchical cluster analysis (HCA).

A critical analysis of the potential for EU Common Agricultural Policy measures to support wild pollinators on farmland.

Agricultural intensification and associated loss of high-quality habitats are key drivers of insect pollinator declines. With the aim of decreasing the environmental impact of agriculture, the 2014 EU Common Agricultural Policy (CAP) defined a set of habitat and landscape features (Ecological Focus Areas: EFAs) farmers could select from as a requirement to receive basic farm payments. To inform the post-2020 CAP, we performed a European-scale evaluation to determine how different EFA options vary in their potential to support insect pollinators under standard and pollinator-friendly management, as well as the extent of farmer uptake.A structured Delphi elicitation process engaged 22 experts from 18 European countries to evaluate EFAs options. By considering life cycle requirements of key pollinating taxa (i.e. bumble bees, solitary bees and hoverflies), each option was evaluated for its potential to provide forage, bee nesting sites and hoverfly larval resources.EFA options varied substantially in the resources they were perceived to provide and their effectiveness varied geographically and temporally. For example, field margins provide relatively good forage throughout the season in Southern and Eastern Europe but lacked early-season forage in Northern and Western Europe. Under standard management, no single EFA option achieved high scores across resource categories and a scarcity of late season forage was perceived.Experts identified substantial opportunities to improve habitat quality by adopting pollinator-friendly management. Improving management alone was, however, unlikely to ensure that all pollinator resource requirements were met. Our analyses suggest that a combination of poor management, differences in the inherent pollinator habitat quality and uptake bias towards catch crops and nitrogen-fixing crops severely limit the potential of EFAs to support pollinators in European agricultural landscapes. Policy Implications. To conserve pollinators and help protect pollination services, our expert elicitation highlights the need to create a variety of interconnected, well-managed habitats that complement each other in the resources they offer. To achieve this the Common Agricultural Policy post-2020 should take a holistic view to implementation that integrates the different delivery vehicles aimed at protecting biodiversity (e.g. enhanced conditionality, eco-schemes and agri-environment and climate measures). To improve habitat quality we recommend an effective monitoring framework with target-orientated indicators and to facilitate the spatial targeting of options collaboration between land managers should be incentivised.

La intensificación agrícola y la consecuente pérdida de hábitats de alta calidad son desencadenantes clave del declive de los insectos polinizadores. Con el objetivo de disminuir el impacto ambiental de la agricultura, la Política Agrícola Común (PAC) de la UE de 2014 definió un conjunto de medidas para hábitats y paisajes (Áreas de Enfoque Ecológico: EFA por sus siglas en inglés) que los agricultores podían seleccionar como requisito para recibir pagos agrícolas básicos. Para informar la reforma de la PAC a partir a 2020, realizamos una evaluación a escala europea para determinar cómo las diferentes opciones de EFA varían en su potencial para asistir a los insectos polinizadores bajo un manejo estándar y amigable con los polinizadores, así como su aceptación por parte de los agricultores.El proceso estructurado de elicitación Delphi para evaluar las opciones de EFA involucró a 22 expertos de 18 países europeos. Se consideraron los requisitos de los diferentes taxones de polinizadores (es decir, abejorros, abejas solitarias y sírfidos) evaluando cada opción por su potencial para proporcionar forraje, sitios de nidificación y recursos para las larvas.Las opciones de EFA variaron sustancialmente en la cantidad de recursos que se percibía que proporcionan y su efectividad vario geográfica y temporalmente. Por ejemplo, los márgenes de cultivos proporcionan un forraje relativamente bueno durante toda la temporada en el sur y el este de Europa, pero carecen de forraje a principios de temporada en el norte y oeste de Europa. Bajo el manejo estándar, ninguna opción de EFA logró puntuaciones altas en todas las categorías de recursos y en general se percibió una escasez de forraje al final de la temporada.Los expertos identificaron oportunidades sustanciales para mejorar la calidad del hábitat mediante la adopción de un manejo amigable con los polinizadores. Sin embargo, mejorar la gestión por sí solo es poco probable que garantice que se cumplan todos los requisitos necesarios para los polinizadores. Nuestro análisis sugiere que una combinación de manejo inadecuado, diferencias de calidad inherentes a los distintos hábitat y el sesgo de aceptación hacia cultivos de cobertura y cultivos que fijan nitrógeno limitan severamente el potencial de los EFA para apoyar a los polinizadores en los paisajes agrícolas europeos. Implicaciones políticas. Para conservar a los polinizadores y ayudar a proteger los servicios de polinización, nuestro estudio destaca la necesidad de crear una variedad de hábitats interconectados y bien administrados que se complementen entre sí en los recursos que ofrecen. Para lograr esto, la PAC post­2020 debe integrar los diferentes vehículos de implementación destinados a proteger la biodiversidad (por ejemplo, condicionalidad mejorada, esquemas ecológicos y medidas agroambientales y climáticas). Para mejorar la calidad del hábitat, recomendamos un marco de monitoreo efectivo con indicadores orientados a objetivos y incentivar la colaboración entre los administradores de las tierras.

L'intensification agricole et la perte associée d'habitats semi­naturels sont les principaux moteurs du déclin des insectes pollinisateurs. Dans l'intention de réduire l'impact environnemental de l'agriculture, la politique agricole commune (PAC) de l'UE de 2014 a défini un ensemble d'habitats et d'éléments paysagers (surfaces d'intérêt écologique: SIE) dans la mise en place ou le respect desquels les agriculteurs pouvaient s'engager comme condition pour bénéficier d'aides économiques européennes (droit au paiement de base). Pour éclairer la PAC post­2020, nous avons évalué à l'échelle européenne et à dire d'expert, d'une part les potentialités des diverses SIE à favoriser les insectes pollinisateurs, via une gestion standard et via une gestion optimisée, et d'autre part l'étendue de l'adoption de ces mesures par les agriculteurs.Un processus structuré d'élaboration et d'agrégation des opinions (méthode Delphi) a fait appel à 22 experts de 18 pays européens pour évaluer les potentialités des diverses SIE. Considérant les traits bioécologiques des principaux taxons pollinisateurs (i.e. bourdons, abeilles solitaires et syrphes), chaque SIE a été évaluée pour son potentiel à fournir des ressources trophiques et des sites de reproduction (sites de nidification pour les bourdons et abeilles, sites de ponte et développement larvaire pour les syrphes).Les SIE différaient considérablement les unes des autres sur les ressources qu'elles étaient censées offrir et leur efficacité variait géographiquement et temporellement. Par exemple, les bords de champ peuvent fournir des ressources trophiques tout au long de l'année en Europe du Sud et de l'Est mais pas en début de saison en Europe du Nord et de l'Ouest. En cas de gestion standard, aucun type de SIE n'atteint de score élevé pour aucun type de ressource, et une période de disette alimentaire survient en fin de saison.Les experts ont mis en évidence de possibles et substantielles améliorations des SIE par le biais de leur gestion optimisée. Cependant, cette seule amélioration ne garantit pas la fourniture de ressources suffisantes aux pollinisateurs des paysages agricoles européens. Pour cela, des habitats spécifiques doivent être favorisés, dont la mise en place ne doit pas être entravée par un choix massif de SIE à base de cultures intermédiaires pièges à nitrates ou fixatrices d'azote. Implications politiques. Pour préserver les pollinisateurs et le service de pollinisation des plantes entomophiles, notre étude souligne la nécessité de créer une diversité d'habitats interconnectés, gérés de façon optimale, qui se complètent mutuellement dans les ressources qu'ils offrent. Pour atteindre cet objectif, la PAC post­2020 doit adopter une vision holistique de la mise en œuvre des différents leviers de protection de la biodiversité (e.g. éco­conditionnalité renforcée, programmes verts ou 'eco­schemes', mesures agro­environnementales et climatiques). Pour réellement améliorer la qualité des habitats, nous recommandons des suivis efficaces de la biodiversité à l'aide d'indicateurs pertinents. Enfin, pour optimiser la disposition spatiale des SIE et leur connectivité, la collaboration entre les différents gestionnaires des espaces agricoles doit être encouragée.

A intensificação agrícola e a perda associada de habitats de elevada qualidade são os principais factores que impulsionam o declínio dos insetos polinizadores. A fim de mitigar o impacto ambiental da agricultura, a Política Agrícola Comum (PAC) da UE, de 2014, definiu um conjunto de atributos ou estruturas do habitat e da paisagem, designadas de Áreas Foco Ecológico (AFEs) que devem ser mantidas pelos agricultores como requisito para obter as ajudas económicas previstas nas medidas agroambientais. No presente trabalho realizamos uma avaliação à escala europeia das diferentes opções destas estruturas, a fim de munir a PAC pós­2020, com informação sobre a importância das AFEs. Estas variam muito quanto ao seu potencial no apoio às populações de polinizadores, de acordo com a extensão da sua aceitação pelos agricultores e das práticas adoptadas por estes na sua gestão, que podem consistir em práticas padrão ou práticas mais amigáveis para os polinizadores.Um processo estruturado, com base na técnica de elicitação de Delphi foi desenvolvido, envolvendo 22 especialistas de 18 países europeus, com o objectivo de avaliar as opções de AFEs previstas na PAC. Esta avaliação levou em consideração os requisitos do ciclo de vida dos taxa dos principais polinizadores, ou seja, as abelhas, as abelhas solitárias e os sirfídeos ou moscas­das­flores. Cada AFE foi avaliada quanto ao seu potencial para fornecer alimento, locais de nidificação, e recursos para as larvas dos sirfídeos.A percepção quanto à eficácia das AFEs como fonte de recursos (alimento) para os polinizadores variou substancialmente, do ponto de vista quer geográfico, quer temporal (época do ano). Por exemplo, a AFE, faixas verdes nas margens do campo são consideradas uma boa fonte de alimento, no sul e leste da Europa, durante todo ano, mas ineficazes, no norte e oeste da Europa, no início do ano. Nenhuma EFA alcançou pontuações elevadas na categoria de recursos (fonte de alimento), quando submetida ao maneio padrão, sendo consideradas ineficientes, na segunda metade do ano.Os especialistas envolvidos identificaram oportunidades de melhoria substancial na qualidade do habitat, através da adopção de práticas de maneio das EFAs mais "amigáveis" para com os polinizadores. No entanto, a melhoria das práticas de maneio das EFAs por si só, dificilmente garantirá todos os requisitos necessários para a manutenção das populações de polinizadores. A nossa avaliação sugere que a combinação de práticas de má gestão (maneio), diferenças inerentes à qualidade do habitat dos polinizadores e o aumento do bias que resulta da utilização de espécies de crescimento rápido ou fixadoras de azoto limitam severamente o papel e potencial destas estruturas na manutenção das populações de polinizadores nas paisagens agrícolas europeias. Implicações políticas. A conservação dos polinizadores ajuda a proteger os serviços de polinização providenciados por estes. O nosso estudo destaca a necessidade de criar uma variedade de habitats interconectados e geridos de forma que se complementem na oferta de recursos (alimento, locais de nidificação e recursos para as larvas) aos polinizadores. Para atingir este objectivo, a PAC pós­2020 deve adoptar uma visão holística na implementação das EFAs, que integre os diferentes programas destinados a protecção da biodiversidade (por exemplo, maior condicionalidade, esquemas ecológicos, e medidas agroambientais e de adaptação climática). Para melhorar a qualidade do habitat, recomendamos uma estrutura de monitorização eficaz suportada por indicadores quantitativos e qualitativos orientados para metas, que permitam facilitar a tomada de decisões direcionadas especificamente para as EFAs, e que a colaboração entre os gestores da terra (agricultores) seja incentivada.

Trace elements bioaccumulation in Porcellionides pruinosus (Brandt, 1833) and related biomarkers of exposure.

In the last years, the increase of anthropogenic activities has led to a progressive pollution of ecosystems. Oniscidean isopods are a group of terrestrial animals known as excellent bio-indicators and bio-accumulators, because of their capacity to accumulate contaminants and to implement appropriate strategies of detoxification. For this study, we collected Porcellionides pruinosus from two different sites in the Corinth region (Greece), a polluted site and a control site, to assess the heavy metal pollution status. We conducted an inductively coupled plasma mass spectrometry (ICP-MS) analysis for the detection of trace elements in animals' hepatopancreas and soil; we performed also immunohistochemistry for detection of metallothioneins 1 (MT1) and heat shock protein 70 (HSP70s). The results suggest a great capacity of the species in trace elements bioaccumulation and the high degree of pollution of the area near Agioi Theodoroi where the metal pollution index (MPI) is threefolds higher than the control site, due to the presence of significantly higher concentration of arsenic, cadmium, cobalt, chromium, manganese, molybdenum, and lead found in hepatopancreas and soil. The environmental stress is confirmed by the expression of the relative biomarkers of exposure MT1 and HSP70. Our study, with a multimarker approach, consents getting a complete analysis of environmental quality and the effect of pollution on organisms and shows for the first time the heavy metals contamination status in certain Greece area. Furthermore, P. pruinosus proved to be an appropriate indicator of pollution in terrestrial ecosystems.

Assessment and management of pesticide pollution at a river basin level part I: Aquatic ecotoxicological quality indices.

During the last decade numerous monitoring programs have been conducted in order to assess pesticide pollution in catchments. This effort has led to the production of large, complex data sets of environmental results making the task of evaluation of the aquatic chemical status more difficult. Furthermore, the evaluation of the chemical status of the water ecosystems is one of the main aspects which should be considered in a River Basin Management Plan. In this study, two indices were developed in order to assess the combined pesticide ecotoxicity to aquatic non-target organisms, the Aquatic Quality Index of Short term Toxicity of Pesticides (AQI ShToxP) and the Aquatic Quality Index of Long term Toxicity of Pesticides (AQI LToxP). These indices were applied to the environmental results obtained from an intensive monitoring study of 302 pesticides in 102 stationary sampling stations located on the surface aquatic network of the Pinios River Basin, in Greece, in 2011 and 2012. The evaluation of the surface water quality was achieved by taking into consideration the frequency and the intensity of exposure of the aquatic organisms to pesticides above the respective ecotoxicological quality objectives such as the acute or chronic term predicted no-effect concentrations derived from risk assessment. Seventy-five pesticides, that have been previously identified as the River Basin Specific Pollutants of Pinios by an environmental and human risk hierarchy exercise, were assessed. It appears, from the implementation of the two indices, that the detected pesticides in the surface aquatic ecosystem of the Pinios River Basin exert significant pressure on the aquatic non-target organisms especially at the chronic effect level. The developed AQI ShToxP and AQI LToxP indices, as well as the proposed quality classification system could be valuable communication and interpretation tools for River Basin Management Plans that can contribute in the restoration of environmental health.

Assessment and management of pesticide pollution at a river basin level part II: Optimization of pesticide monitoring networks on surface aquatic ecosystems by data analysis methods.

The high cost of extensive pesticide monitoring studies, required for the protection of water resources, and the necessity of early identification of environmental threats, highlighted the need for prioritization of pesticides and sampling sites to be monitored. The aim of this study was to develop an optimum surface water monitoring network at a catchment scale including only the sites of a catchment vulnerable to pesticide pollution. The identification of sampling sites vulnerable to pesticide pollution (VPS) was based on the data of an intensive monitoring survey of 302 pesticides in 102 stationary sampling sites located on the surface water network of a river basin. In the proposed methodology the left-censored data of the analytical results derived from the above mentioned monitoring campaign were included in the statistical analyses by transforming all the raw data into categorical variables and arranging them in ordinal scales based on ecotoxicological thresholds derived from pesticide toxicity tests on aquatic non-target organisms. The categorized data were subjected to Categorical Principal Component Analysis with Optimal Scaling. For the identification of the VPS, the Squared Mahalanobis Distance criterion was applied on the extracted values (scores) of the significant principal components. With this methodology a 46% reduction in the number of the monitoring stations was achieved. This approach will be valuable in establishing more cost effective monitoring schemes in the future in other basins and in developing targeted measures to eliminate or limit the effect of critical pollution sources in surface aquatic systems. Moreover, by applying the proposed methodology, historical monitoring data can be used to initiate more efficient pesticide monitoring campaigns in the future.

Variability of pesticide residues in eggplant units collected from a field trial and marketplaces in Greece.

BACKGROUND: Variability of pesticide residues among food items is very important when assessing the risks and food safety for the consumers. Therefore, the present study was undertaken to estimate the unit-to-unit residue variability factors for eggplant. RESULTS: In total, 120 samples from a trial field and 142 samples from different marketplaces in Thessaloniki, Greece, were collected to estimate the variability of pesticide residues in eggplant units. They were extracted by the QuEChERS method and the residues were determined by LC-MS/MS. For the field samples, the unit-to-unit variability factors (VFs) obtained for cypermethrin and deltamethrin residues were 2.54 and 2.51, respectively. The mean residue levels of both pesticides were higher in the composite samples than in the individual samples. The average VFs for the marketplace samples was 3.89. The eggplant units exposed to pesticides were higher in residues than the non-exposed units. CONCLUSION: The variability factors obtained in the marketplace samples were higher than those in the samples collected from the field trial. A default VF value of 3 for field trials is appropriate for use when assessing the acute dietary intake but a VF for the marketplace samples should be reconsidered with a larger data. © 2017 Society of Chemical Industry.

Pesticides in the rivers and streams of two river basins in northern Greece.

The pollution caused by pesticides, and their ecotoxicological implications were investigated in water samples from the Strymonas and Nestos river basins (Northern Greece). Chlorpyrifos was the most frequently detected pesticide in both basins (42 and 37% in the Strymonas and Nestos basins, respectively), followed by fluometuron and terbuthylazine (25 and 12%, Strymonas), and bentazone and boscalid (24 and 10%, Nestos). The Annual Average and the Maximum Allowable Concentration of Environmental Quality Standards set in European Union Directives were exceeded in several cases by alphamethrin and chlorpyrifos. Risk Quotient assessment revealed significant ecological risk towards the aquatic organisms in over 20% of the water samples. Insecticides (mostly pyrethroids and organophosphosphates) contributed more in the ecotoxicological risk than herbicides and fungicides. The three main rivers in the current study (Strymonas, Aggitis, Nestos) exhibited similar sum of RQs indicating that aquatic life in all three of them was at the same risk level. However, the sums of RQs were higher in the various streams monitored than the three rivers.

Environmental and human risk hierarchy of pesticides: A prioritization method, based on monitoring, hazard assessment and environmental fate.

A pesticide prioritization approach was developed and implemented in the Pinios River Basin of Central Greece. It takes under consideration the Level of Environmental Risk containing information on the frequency of occurrence of pesticides above environmental thresholds, the intensity of this occurrence and the spatial distribution as well as information about the fate and behavior of pesticides in the environment and the potential to have adverse impact on humans' health. Original 3-year monitoring data from 102 Stationary Sampling Sites located on rivers and their tributaries, reservoirs, streams and irrigation/drainage canals giving rise to a collection of 2382 water samples resulting in 7088 data sets, were included in this integrated prioritization study. Among 302 monitored active ingredients, 119 were detected at least once and the concentrations found in the aquatic systems for 41% of compounds were higher than the respective lowest Predicted Non-Effect Concentration (PNEC) values. Sixteen and 5 pesticides were found with risk ratios (MECmax/PNEC) above 10 (high concern) and 100 (very high concern), respectively. However, pesticides with maximum Measured Environmental Concentration (MECmax) values exceeding by 1000 times the respective lowest PNEC values were also found which were considered of extremely high concern; in the latter group were included prometryn, chlorpyrifos, diazinon, λ-cyhalothrin, cypermethrin, α-cypermethrin deltamethrin, ethalfluralin and phosmet. The sensitivity of the analytical methods used in the monitoring study was considered inadequate to meet the toxicological endpoints for 32 pesticides. The widest distribution of occurrence in the Stationary Sampling Sites of the monitoring program was found for the pesticides, prometryn, fluometuron, terbuthylazine, S-metolachlor, chlorpyrifos, diphenylamine, acetochlor, alachlor, 2,4-D, etridiazole, imidacloprid and lindane (γ-ΗCH). Among the 27 priority pesticides included in the Directive 2013/39/EU, in the present study 13 pesticides were considered as candidates for River Basin Specific Pollutants (RBSP) for the River Basin of Pinios. Among the 30 pesticide specific pollutants that were established by the Greek authorities, in the present study only 6 were considered as candidates for RBSP for the river basin of Pinios. As a result of the implementation of the prioritization approach developed in this study a total of 71 pesticides were identified as being RBSP for the river basin of Pinios. The higher Level of Environmental Risk was found to be exerted by the organophosphorus insecticide chlorpyrifos, followed by lindane and prometryn. The present study provides background information for important decisions to be made concerning the selection of pesticides which should be included in the target analyte list of new monitoring and screening programs of surface water quality in the Pinios River Basin. In addition, the prioritization approach proposed here can be useful for the development of River Basin Management Plans.

Pesticides in the surface waters of Lake Vistonis Basin, Greece: Occurrence and environmental risk assessment.

A study was undertaken for the evaluation of the pesticide pollution caused by the agricultural activities in the basin of Lake Vistonis, Greece during the years 2010-2012. Water samples were collected from Lake Vistonis, four major rivers and various small streams and agriculture drainage canals. The concentration of 302 compounds was determined after solid-phase extraction of the water samples and subsequent LC-MS/MS and GC-MS/MS analysis of the extracts. Overall, herbicides were the most frequently detected pesticides (57%), followed by insecticides (28%) and fungicides (14%). In Lake Vistonis 11 pesticides were detected. Specifically, fluometuron was detected in the 75% of the samples (maximum concentration 0.088 µg/L) whereas lambda-cyhalothrin was detected in all the samples of spring 2011 and alphamethrin in all the samples of spring 2012 (maximum concentration 0.041 and 0.168 µg/L, respectively). In the rivers and drainage canals 68 pesticides were detected. Specifically, fluometuron was detected in the 53% of the samples (maximum concentration 317.6 µg/L) followed by chlorpyrifos and prometryn (16 and 13% of the samples respectively). An environmental risk assessment was performed by employing the Risk Quotient (RQ) method. The risk assessment revealed that at least one pesticide concentration led to a RQ>1 in 20% of the samples. In Lake Vistonis, alphamethrin and lambda-cyhalothrin concentrations resulted in RQ>1, whereas in the other water bodies this was mainly the result of chlorpyrifos-methyl and alphamethrin exposure. In contrast, herbicide and fungicide concentrations contributed substantially less to environmental risks.