Insights into the multitrophic interactions between the biocontrol agent Bacillus subtilis MBI 600, the pathogen Botrytis cinerea and their plant host

Botrytis cinerea is a plant pathogen causing the gray mold disease in a plethora of host plants. The control of the disease is based mostly on chemical pesticides, which are responsible for environmental pollution, while they also pose risks for human health. Furthermore, B. cinerea resistant isolates have been identified against many fungicide groups, making the control of this disease challenging. The application of biocontrol agents can be a possible solution, but requires deep understanding of the molecular mechanisms in order to be effective. In this study, we investigated the multitrophic interactions between the biocontrol agent Bacillus subtilis MBI 600, a new commercialized biopesticide, the pathogen B. cinerea and their plant host. Our analysis showed that this biocontrol agent reduced B. cinerea mycelial growth in vitro, and was able to suppress the disease incidence on cucumber plants. Moreover, treatment with B. subtilis led to induction of genes involved in plant immunity. RNAseq analysis of B. cinerea transcriptome upon exposure to bacterial secretome, showed that genes coding for MFS and ABC transporters were highly induced. Deletion of the Bcmfs1 MFS transporter gene, using a CRISP/Cas9 editing method, affected its virulence and the tolerance of B. cinerea to bacterial secondary metabolites. These findings suggest that specific detoxification transporters are involved in these interactions, with crucial role in different aspects of B. cinerea physiology

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

Initial vehicle design with stability criteria

In order to define the handling properties of a vehicle at the very first design stages many assumptions and simplifications are necessary. The most typical approach is by using simplified numerical methods for predicting the vehicle handling characteristics such as the one track vehicle model. In most cases this model doesn’t allows for successful choice of the basic geometrical dimensions of the suspension or the steering mechanism. For this reason a new more complete procedure is needed in order to choose the basic geometrical dimensions and properties of a vehicle at the first design stage. For the examination of the above characteristics a new mathematical model was developed and the stability and the directional behavior of a vehicle negotiating a turn at constant forward speed were analyzed. The value of the presented model is that the influence of both suspension and steering mechanisms to the final vehicle handling properties can be predicted with high accuracy without the need of their geometrical description. Finally, with the vehicle Formula ART 7 as basis parametrical analysis were made in order to examine the influence of the geometrical dimensions to the stability, directional control and handling properties of the vehicles.Για τον προσδιορισμό της οδικής συμπεριφοράς ενός οχήματος κατά τα πρώτα στάδια του σχεδιασμού του, απαιτείται πλήθος παραδοχών και απλοποιήσεων. Η συνηθέστερη προσέγγιση γίνεται με τη βοήθεια απλοποιημένων υπολογιστικών διαδικασιών όπως αυτής του μοντέλου ενός ίχνους. Σε πολλές περιπτώσεις όμως η προσέγγιση αυτή δεν επιτρέπει την επιτυχημένη εκλογή των βασικών γεωμετρικών διαστάσεων της ανάρτησης και του συστήματος διεύθυνσης. Γι’ αυτό υπάρχει ανάγκη να αναπτυχθεί μία πιο πλήρης διαδικασία για την εκλογή των βασικών γεωμετρικών μεγεθών που χαρακτηρίζουν την οδική συμπεριφορά ενός οχήματος. Για τη διερεύνηση της επίδρασης των παραπάνω μεγεθών στην οδική συμπεριφορά ενός οχήματος ο κ. Καραογλανίδης ανέπτυξε ένα νέο μοντέλο υπολογισμού των συντελεστών ευστάθειας και κατευθυντικότητας ενός οχήματος τεσσάρων τροχών που μπορεί να εφαρμοστεί από το αρχικό στάδιο του σχεδιασμού του. Η αξία της προτεινόμενης διαδικασίας έγκειται στο γεγονός ότι τόσο η επίδραση του μηχανισμού της ανάρτησης όσο και του συστήματος διεύθυνσης μπορούν να προσδιορισθούν με επαρκή ακρίβεια χωρίς την ανάγκη της πλήρους γεωμετρικής περιγραφής τους. Σε επόμενο στάδιο της εργασίας του ο υποψήφιος υπολόγισε και σχεδίασε την ανάρτηση και το σύστημα διεύθυνσης του αγωνιστικού οχήματος Formula ART 7 του οποίου μέρος κατασκευάστηκε στο Εργαστήριο Στοιχείων Μηχανών και Μηχανολογικού Σχεδιασμού. Το όχημα αυτό επιτρέπει την μελέτη της επίδρασης διαφόρων γεωμετρικών παραμέτρων στην οδική συμπεριφορά του μέσω ρυθμίσεων του μηχανισμού της ανάρτησης και του συστήματος διεύθυνσης. Στη συνέχεια, έχοντας ως βάση σύγκρισης τα γεωμετρικά και τεχνικά χαρακτηριστικά του αγωνιστικού οχήματος ART 7, ο υποψήφιος επιβεβαίωσε την ακρίβεια των υπολογισμών συγκρίνοντας τους με τα αποτελέσματα που προκύπτουν με χρήση άλλων γνωστών μοντέλων. Συγκεκριμένα, επιλέχθηκαν το μοντέλο ενός ίχνους και η εφαρμογή ADAMS της MSC. Στο τελευταίο μέρος της εργασίας του ο υποψήφιος χρησιμοποιώντας την προαναφερθείσα μεθοδολογία έκανε συγκριτική ανάλυση οχημάτων με μεταβλητές παραμέτρους και παρουσίασε διαγράμματα μεταβολής των συντελεστών ευστάθειας και κατευθυντικότητας. Τα διαγράμματα αυτά μπορούν να λειτουργήσουν ως συστάσεις για την επιλογή των βασικών γεωμετρικών και κατασκευαστικών μεγεθών των οχημάτων κατά το στάδιο του αρχικού σχεδιασμού

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

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

Under Pressure: A Comparative Study of Botrytis cinerea Populations from Conventional and Organic Farms in Cyprus and Greece

The highly heterogeneous nature of Botrytis cinerea provides adaptive benefits to variable environmental regimes. Disentangling pathogen population structure in anthropogenic agroecosystems is crucial to designing more effective management schemes. Herein, we studied how evolutionary forces exerted in different farming systems, in terms of agrochemicals-input, shape B. cinerea populations. In total, 360 B. cinerea isolates were collected from conventional and organic, strawberry and tomato farms in Cyprus and Greece. The occurrence and frequency of sensitivities to seven botryticides were estimated. Results highlighted widespread fungicide resistance in conventional farms since only 15.5% of the isolates were sensitive. A considerable frequency of fungicide-resistant isolates was also detected in the organic farms (14.9%). High resistance frequencies were observed for boscalid (67.7%), pyraclostrobin (67.3%), cyprodinil (65.9%), and thiophanate-methyl (61.4%) in conventional farms, while high levels of multiple fungicide resistance were also evident. Furthermore, B. cinerea isolates were genotyped using a set of seven microsatellite markers (simple sequence repeat [SSR] markers). Index of association analyses (Ia and rBarD) suggest asexual reproduction of the populations, even though the mating-type idiomorphs were equally distributed, indicating frequency-dependent selection. Fungicide resistance was correlated with farming systems across countries and crops, while SSRs were able to detect population structure associated with resistance to thiophanate-methyl, pyraclostrobin, boscalid, and cyprodinil. The expected heterozygosity in organic farms was significantly higher than in conventional, suggesting the absence of selective pressure that may change the allelic abundance in organic farms. However, genetic variance among strawberry and tomato populations was high, ranking host specificity higher than other selection forces studied

Root Transcriptional and Metabolic Dynamics Induced by the Plant Growth Promoting Rhizobacterium (PGPR) Bacillus subtilis Mbi600 on Cucumber Plants

Bacillus subtilis MBI600 is a commercialized plant growth-promoting bacterial species used as a biocontrol agent in many crops, controlling various plant pathogens via direct or indirect mechanisms. In the present study, a detailed transcriptomic analysis of cucumber roots upon response to the Bs MBI600 strain is provided. Differentially expressed genes (DEGs) analysis showed altered gene expression in more than 1000 genes at 24 and 48 h post-application of Bs MBI600. Bs MBI600 induces genes involved in ISR and SAR signaling. In addition, genes involved in phytohormone production and nutrient availability showed an upregulation pattern, justifying the plant growth promotion. Biocontrol ability of Bs MBI600 seems also to be related to the activation of defense-related genes, such as peroxidase, endo-1,3(4)-beta-glucanase, PR-4, and thaumatin-like. Moreover, KEGG enriched results showed that differentially expressed genes were classified into biocontrol-related pathways. To further investigate the plant's response to the presence of PGPR, a profile of polar metabolites of cucumber treated with Bs MBI600 was performed and compared to that of untreated plants. The results of the current study gave insights into the mechanisms deployed by this biocontrol agent to promote plant resistance, helping to understand the molecular interactions in this system