Multiple and multidrug resistance in Botrytis cinerea: molecular mechanisms of MLR/MDR strains in Greece and effects of co-existence of different resistance mechanisms on fungicide sensitivity

Botrytis cinerea is a high-risk pathogen for fungicide resistance development. Within the fungal populations, strains have developed multiple mutations in different target genes leading to multiple resistance (MLR) or mutations associated with overexpression of efflux transporters leading to multidrug resistance (MDR). These types of resistance are a major threat, and their successful management is a major challenge. The current study was initiated to a) determine frequencies of MLR/MDR strains in populations originating from several crops, b) identify the types of MDR that occur in Greece, and c) determine interactions between MLR and MDR at the level of sensitivity to botryticides. The frequencies of MLR/MDR phenotypes were determined in 515 isolates subjected to bioassays using discriminatory concentrations of thiophanate-methyl, iprodione, cyprodinil, fenhexamid, boscalid, fluopyram, fludioxonil, pyraclostrobin, and tolnaftate. Interestingly, 7.8% and 31.3% of isolates from strawberry and rootstock seedlings were resistant to every single fungicide class, while MDR phenotypes from strawberries, rootstocks, and tomatoes accounted for 26%, 87%, and 13.4%, respectively. The MLR and MDR isolates were further molecularly analyzed regarding genes erg27, sdhB, Bcpos5, and Mrr1, responsible for resistance to fenhexamid, boscalid and fluopyram, cyprodinil, and MDR, respectively. The different mutations’ presence was determined along with a new mutation in Mrr1 leading to MDR. MDR isolates were characterized as MDR1 or MDR1h based on the presence of a 3-bp deletion in Mrr1. MDR1h was predominant in isolates from rootstocks and MDR1 from tomatoes and strawberries, whereas the most frequent target-site mutations were F412S (erg27), H272R (sdhB), and L412F (Bcpos5). To determine whether the accumulation of target-site mutations along with MDR mutations exhibits an additive effect concerning fungicide resistance, the sensitivity of isolates possessing the predominant target-site mutations was calculated in both the presence and the absence of MDR-associated mutations. EC50 in cyprodinil and boscalid increased to about twofold in the presence of MDR mutations, while there was no difference for fenhexamid. In conclusion, MLR/MDR frequencies are notably high in heavily treated crops in Greece, and the combination of MLR and MDR mutations leads to even higher fungicide resistance levels, highlighting the importance of resistance management

The house in prepalatial Crete: architectural form, constitution and social dimension

The present thesis examined the domestic architecture of Prepalatial Crete. The aims were three. First, a new synthesis of the evolution of Prepalatial architecture. Second, the determination of its social role. Third, to evaluate its contribution to the palatial phenomenon. The reason for this was the absence of a study that would consider the Prepalatial architectural remains. The available architectural material, which has been greatly enriched following recent excavation research, had not been evaluated as a whole in the study of the social and historical development of the island during the Prepalatial period and towards the palaces. The present thesis considered it appropriate to distance itself from the typological and morphological approach because it had not yet borne fruit. It considered that Prepalatial architectural remains can be better approached from a design point of view because of, firstly, their considerable morphological heterogeneity and, secondly, the fragmentary nature that characterizes them in many cases. This thesis has demonstrated that Prepalatial architecture can be composed of buildings of varying form that at first level appear different, but that are nevertheless made up of individual spaces with common organizational principles. Furthermore, it was found to possess clear characteristics in the temporal evolution and geographical dispersion of these organizing principles. In the end, Prepalatial architecture is another form of material culture of Prepalatial Crete. This statement does not simply mean a set of human creations with common morphological characteristics, but another material medium and at the same time the result of social interaction in a specific geographical, temporal and historical context. Furthermore, the existence and significance of a Prepalatial domestic field as a whole was brought back into focus. The main conclusion of this thesis was that in Prepalatial Crete there was a common cultural consensus in the organization of space into the houses and ultimately in the design of the settlements. This view to some extent modifies the current image of the island as a set of fragmented communities. Finally, the Minoan palace can be placed in the genealogy of Minoan architecture after the Prepalatial communities, the architectural environment of which constituted a space for social interaction and ultimately the strengthening of a 'Minoan ethos'.Η παρούσα διατριβή εξέτασε την οικιστική αρχιτεκτονική της προανακτορικής Κρήτης. Οι στόχοι ήταν τρεις. Πρώτον, μία νέα σύνθεση της εξέλιξης της προανακτορικής αρχιτεκτονικής. Δεύτερον, ο καθορισμός του κοινωνικού ρόλου της. Τρίτον, η αξιολόγηση της συμβολής της στο ανακτορικό φαινόμενο. Αφορμή αποτέλεσε η απουσία μίας μελέτης η οποία να συνεξετάζει τα προανακτορικά αρχιτεκτονικά κατάλοιπα. Το διαθέσιμο αρχιτεκτονικό υλικό, το οποίο ύστερα και από τις πρόσφατες ανασκαφικές έρευνες εμπλουτίστηκε σε μεγάλο βαθμό, δεν είχε αξιολογηθεί συνολικά στη μελέτη για την κοινωνική και ιστορική εξέλιξη του νησιού κατά την Προανακτορική περίοδο και την πορεία προς τα ανάκτορα. Η παρούσα διατριβή έκρινε σκόπιμη την αποστασιοποίηση από την τυπολογική και μορφολογική προσέγγιση διότι μέχρι σήμερα δεν είχε αποδώσει καρπούς. Θεώρησε ότι τα προανακτορικά αρχιτεκτονικά κατάλοιπα μπορούν να προσεγγιστούν καλύτερα από άποψη σχεδιασμού εξαιτίας πρώτον της σημαντικής μορφολογικής ετερογένειάς τους και δεύτερον της αποσπασματικότητας που τα χαρακτηρίζει σε αρκετές περιπτώσεις. Η παρούσα διατριβή απέδειξε ότι η προανακτορική αρχιτεκτονική μπορεί να αποτελείται από κτήρια ποικίλης μορφής που σε πρώτο επίπεδο δείχνουν διαφορετικά, ωστόσο αυτά συγκροτούνται από επιμέρους χώρους με κοινές οργανωτικές αρχές. Επιπλέον, διαπιστώθηκε ότι διέθετε σαφή χαρακτηριστικά στη διαχρονική εξέλιξη και τη γεωγραφική διασπορά αυτών των οργανωτικών αρχών της. Εν τέλει, η προανακτορική αρχιτεκτονική αποτελεί μία ακόμα διακριτή μορφή υλικού πολιτισμού της προανακτορικής Κρήτης. Με τη διαπίστωση αυτή δεν εννοείται απλώς ένα σύνολο ανθρώπινων δημιουργημάτων με κοινά μορφολογικά χαρακτηριστικά, αλλά ένα ακόμη υλικό μέσο και ταυτόχρονα αποτέλεσμα της κοινωνικής διάδρασης σε συγκεκριμένο γεωγραφικό, χρονικό και ιστορικό πλαίσιο. Επιπλέον, τέθηκε ξανά στο επίκεντρο η ύπαρξη και η σημασία ενός προανακτορικού οικιστικού πεδίου συνολικά. Κύριο συμπέρασμα της παρούσας διατριβής ήταν ότι στην προανακτορική Κρήτη υπήρχε κοινή πολιτισμική συναντίληψη στην οργάνωση του χώρου των οικημάτων και εν τέλει στον γενικότερο σχεδιασμό των οικισμών. Η άποψη αυτή ως ένα βαθμό τροποποιεί την ισχύουσα εικόνα του νησιού ως συνόλου κατακερματισμένων κοινοτήτων. Τέλος, το μινωικό ανάκτορο μπορεί να τοποθετηθεί στη γενεαλογία της μινωικής αρχιτεκτονικής ύστερα από τις προανακτορικές κοινότητες, το αρχιτεκτονικό περιβάλλον των οποίων, συνιστούσε χώρο κοινωνικής συναναστροφής και τελικά ενδυνάμωσης ενός «μινωικού έθους»

Table_2_Multiple and multidrug resistance in Botrytis cinerea: molecular mechanisms of MLR/MDR strains in Greece and effects of co-existence of different resistance mechanisms on fungicide sensitivity.docx

Botrytis cinerea is a high-risk pathogen for fungicide resistance development. Within the fungal populations, strains have developed multiple mutations in different target genes leading to multiple resistance (MLR) or mutations associated with overexpression of efflux transporters leading to multidrug resistance (MDR). These types of resistance are a major threat, and their successful management is a major challenge. The current study was initiated to a) determine frequencies of MLR/MDR strains in populations originating from several crops, b) identify the types of MDR that occur in Greece, and c) determine interactions between MLR and MDR at the level of sensitivity to botryticides. The frequencies of MLR/MDR phenotypes were determined in 515 isolates subjected to bioassays using discriminatory concentrations of thiophanate-methyl, iprodione, cyprodinil, fenhexamid, boscalid, fluopyram, fludioxonil, pyraclostrobin, and tolnaftate. Interestingly, 7.8% and 31.3% of isolates from strawberry and rootstock seedlings were resistant to every single fungicide class, while MDR phenotypes from strawberries, rootstocks, and tomatoes accounted for 26%, 87%, and 13.4%, respectively. The MLR and MDR isolates were further molecularly analyzed regarding genes erg27, sdhB, Bcpos5, and Mrr1, responsible for resistance to fenhexamid, boscalid and fluopyram, cyprodinil, and MDR, respectively. The different mutations’ presence was determined along with a new mutation in Mrr1 leading to MDR. MDR isolates were characterized as MDR1 or MDR1h based on the presence of a 3-bp deletion in Mrr1. MDR1h was predominant in isolates from rootstocks and MDR1 from tomatoes and strawberries, whereas the most frequent target-site mutations were F412S (erg27), H272R (sdhB), and L412F (Bcpos5). To determine whether the accumulation of target-site mutations along with MDR mutations exhibits an additive effect concerning fungicide resistance, the sensitivity of isolates possessing the predominant target-site mutations was calculated in both the presence and the absence of MDR-associated mutations. EC50 in cyprodinil and boscalid increased to about twofold in the presence of MDR mutations, while there was no difference for fenhexamid. In conclusion, MLR/MDR frequencies are notably high in heavily treated crops in Greece, and the combination of MLR and MDR mutations leads to even higher fungicide resistance levels, highlighting the importance of resistance management.</p

Table_1_Multiple and multidrug resistance in Botrytis cinerea: molecular mechanisms of MLR/MDR strains in Greece and effects of co-existence of different resistance mechanisms on fungicide sensitivity.docx

Botrytis cinerea is a high-risk pathogen for fungicide resistance development. Within the fungal populations, strains have developed multiple mutations in different target genes leading to multiple resistance (MLR) or mutations associated with overexpression of efflux transporters leading to multidrug resistance (MDR). These types of resistance are a major threat, and their successful management is a major challenge. The current study was initiated to a) determine frequencies of MLR/MDR strains in populations originating from several crops, b) identify the types of MDR that occur in Greece, and c) determine interactions between MLR and MDR at the level of sensitivity to botryticides. The frequencies of MLR/MDR phenotypes were determined in 515 isolates subjected to bioassays using discriminatory concentrations of thiophanate-methyl, iprodione, cyprodinil, fenhexamid, boscalid, fluopyram, fludioxonil, pyraclostrobin, and tolnaftate. Interestingly, 7.8% and 31.3% of isolates from strawberry and rootstock seedlings were resistant to every single fungicide class, while MDR phenotypes from strawberries, rootstocks, and tomatoes accounted for 26%, 87%, and 13.4%, respectively. The MLR and MDR isolates were further molecularly analyzed regarding genes erg27, sdhB, Bcpos5, and Mrr1, responsible for resistance to fenhexamid, boscalid and fluopyram, cyprodinil, and MDR, respectively. The different mutations’ presence was determined along with a new mutation in Mrr1 leading to MDR. MDR isolates were characterized as MDR1 or MDR1h based on the presence of a 3-bp deletion in Mrr1. MDR1h was predominant in isolates from rootstocks and MDR1 from tomatoes and strawberries, whereas the most frequent target-site mutations were F412S (erg27), H272R (sdhB), and L412F (Bcpos5). To determine whether the accumulation of target-site mutations along with MDR mutations exhibits an additive effect concerning fungicide resistance, the sensitivity of isolates possessing the predominant target-site mutations was calculated in both the presence and the absence of MDR-associated mutations. EC50 in cyprodinil and boscalid increased to about twofold in the presence of MDR mutations, while there was no difference for fenhexamid. In conclusion, MLR/MDR frequencies are notably high in heavily treated crops in Greece, and the combination of MLR and MDR mutations leads to even higher fungicide resistance levels, highlighting the importance of resistance management.</p

Image_1_Multiple and multidrug resistance in Botrytis cinerea: molecular mechanisms of MLR/MDR strains in Greece and effects of co-existence of different resistance mechanisms on fungicide sensitivity.jpeg

Botrytis cinerea is a high-risk pathogen for fungicide resistance development. Within the fungal populations, strains have developed multiple mutations in different target genes leading to multiple resistance (MLR) or mutations associated with overexpression of efflux transporters leading to multidrug resistance (MDR). These types of resistance are a major threat, and their successful management is a major challenge. The current study was initiated to a) determine frequencies of MLR/MDR strains in populations originating from several crops, b) identify the types of MDR that occur in Greece, and c) determine interactions between MLR and MDR at the level of sensitivity to botryticides. The frequencies of MLR/MDR phenotypes were determined in 515 isolates subjected to bioassays using discriminatory concentrations of thiophanate-methyl, iprodione, cyprodinil, fenhexamid, boscalid, fluopyram, fludioxonil, pyraclostrobin, and tolnaftate. Interestingly, 7.8% and 31.3% of isolates from strawberry and rootstock seedlings were resistant to every single fungicide class, while MDR phenotypes from strawberries, rootstocks, and tomatoes accounted for 26%, 87%, and 13.4%, respectively. The MLR and MDR isolates were further molecularly analyzed regarding genes erg27, sdhB, Bcpos5, and Mrr1, responsible for resistance to fenhexamid, boscalid and fluopyram, cyprodinil, and MDR, respectively. The different mutations’ presence was determined along with a new mutation in Mrr1 leading to MDR. MDR isolates were characterized as MDR1 or MDR1h based on the presence of a 3-bp deletion in Mrr1. MDR1h was predominant in isolates from rootstocks and MDR1 from tomatoes and strawberries, whereas the most frequent target-site mutations were F412S (erg27), H272R (sdhB), and L412F (Bcpos5). To determine whether the accumulation of target-site mutations along with MDR mutations exhibits an additive effect concerning fungicide resistance, the sensitivity of isolates possessing the predominant target-site mutations was calculated in both the presence and the absence of MDR-associated mutations. EC50 in cyprodinil and boscalid increased to about twofold in the presence of MDR mutations, while there was no difference for fenhexamid. In conclusion, MLR/MDR frequencies are notably high in heavily treated crops in Greece, and the combination of MLR and MDR mutations leads to even higher fungicide resistance levels, highlighting the importance of resistance management.</p

Challenges for Sustained Observing and Forecasting Systems in the Mediterranean Sea

The Mediterranean community represented in this paper is the result of more than 30 years of EU and nationally funded coordination, which has led to key contributions in science concepts and operational initiatives. Together with the establishment of operational services, the community has coordinated with universities, research centers, research infrastructures and private companies to implement advanced multi-platform and integrated observing and forecasting systems that facilitate the advancement of operational services, scientific achievements and mission-oriented innovation. Thus, the community can respond to societal challenges and stakeholders needs, developing a variety of fit-for-purpose services such as the Copernicus Marine Service. The combination of state-of-the-art observations and forecasting provides new opportunities for downstream services in response to the needs of the heavily populated Mediterranean coastal areas and to climate change. The challenge over the next decade is to sustain ocean observations within the research community, to monitor the variability at small scales, e.g., the mesoscale/submesoscale, to resolve the sub-basin/seasonal and inter-annual variability in the circulation, and thus establish the decadal variability, understand and correct the model-associated biases and to enhance model-data integration and ensemble forecasting for uncertainty estimation. Better knowledge and understanding of the level of Mediterranean variability will enable a subsequent evaluation of the impacts and mitigation of the effect of human activities and climate change on the biodiversity and the ecosystem, which will support environmental assessments and decisions. Further challenges include extending the science-based added-value products into societal relevant downstream services and engaging with communities to build initiatives that will contribute to the 2030 Agenda and more specifically to SDG14 and the UN's Decade of Ocean Science for sustainable development, by this contributing to bridge the science-policy gap. The Mediterranean observing and forecasting capacity was built on the basis of community best practices in monitoring and modeling, and can serve as a basis for the development of an integrated global ocean observing system