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

Characterization of laboratory and real driving emissions of individual Euro 6 light-duty vehicles – Fresh particles and secondary aerosol formation

Emissions from passenger cars are one of major sources that deteriorate urban air quality. This study presents characterization of real-drive emissions from three Euro 6 emission level passenger cars (two gasoline and one diesel) in terms of fresh particles and secondary aerosol formation. The gasoline vehicles were also characterized by chassis dynamometer studies. In the real-drive study, the particle number emissions during regular driving were 1.1–12.7 times greater than observed in the laboratory tests (4.8 times greater on average), which may be caused by more effective nucleation process when diluted by real polluted and humid ambient air. However, the emission factors measured in laboratory were still much higher than the regulatory value of 6 × 10^(11) particles km^(−1). The higher emission factors measured here result probably from the fact that the regulatory limit considers only non-volatile particles larger than 23 nm, whereas here, all particles (also volatile) larger than 3 nm were measured. Secondary aerosol formation potential was the highest after a vehicle cold start when most of the secondary mass was organics. After the cold start, the relative contributions of ammonium, sulfate and nitrate increased. Using a novel approach to study secondary aerosol formation under real-drive conditions with the chase method resulted mostly in emission factors below detection limit, which was not in disagreement with the laboratory findings

HELIOS/SICRIT/mass spectrometry for analysis of aerosols in engine exhaust

Current legislations typically characterize systems of aerosols, such as from vehicle exhaust, primarily by number concentration and size distributions. While potential health threats have a dependence on the particle size, the chemical composition of particles, including the volatile and semi-volatile components adsorbed onto nonvolatile particle cores present at roadside and urban settings, is important in understanding the impact of exhaust particles on health. To date, the only tools suitable for an online in-depth chemical aerosol characterization are aerosol mass spectrometers, which are typically composed of complex and cost intensive instrumentation. We present a new analytical system, which combines a novel inexpensive infrared-radiation-based evaporation system (HELIOS) with a commercially available highly efficient atmospheric ionization source (SICRIT) connected to a rather low-price ion-trap mass spectrometer. Our inexpensive, robust and mobile aerosol characterization HELIOS/SICRIT/Mass Spectrometry system enables highly sensitive chemical analysis of particle-associated volatile substances. We validate the HELIOS/SICRIT/Mass Spectrometry system in laboratory experiments with coated particles generated under controlled conditions, and show that the system is capable of identification of combustion-generated polycyclic aromatic hydrocarbons and relative quantification of individual chemical species adsorbed on particle surfaces. We then employ our system to analyze real-world vehicle engine exhaust aerosol and show through time-resolved measurements with high time resolution (<10 s) that the chemical composition of the particles changes during different parts of an engine test cycle.acceptedVersionPeer reviewe

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&rsquo;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

Characterization of Bacillus subtilis MBI600 as biocontrol agent against plant pathogens and plant growth promoter

The use of beneficial microbes in modern agriculture is no longer considered an exception but a necessity in all cultivation strategies. Beneficial rhizobacteria are an important part of commercial products because of their ability to promote plant growth and their effectiveness against plant pathogenic microbes. Bacteria belonging to the genus Bacillus constitute the majority of beneficial microorganisms in the market. The commercial formulation Serifel® contains the biological agent Bacillus subtilis MBI600 (Bs MBI600), and in the present dissertation a systematic recording of the mechanisms of action of this beneficial microorganism is presented. In order to better understand the mechanisms of action of Bs MBI600, its whole genome was sequenced with Illumina HiSeq X. The results of the genomic analysis revealed genes associated with the production of substances that act as communication signals with host-plants, leading to successful root colonization. Furthermore, genes related to the ability of biological agents to increase the availability of plant nutrients, as well as the production of phytohormones that lead to the promotion of plant growth were also identified. Finally, genes involved in the production of antimicrobial compounds were identified in the Bs MBI600 genome.The colonization ability of Bs MBI600 on tomato and cucumber roots was studied in different soil substrates, using a yellow fluorescence protein (yfp)- transformed strain of Bs MBI600. Bacterial cells of the strain were naturally transformed by the introduction of the fluorescence protein. Transformed strains were studied using an inverted fluorescence microscope, and root surface colonization was recorded 48 hours after treatment. Subsequently, the reproducibility on different soil substrates (hydroponics, 2 soil types and sterile gnotobiotic system) was recorded. Bs MBI600, was found able to colonize the root system of both hosts in all the 4 substrates. However, significant variations were observed in the duration and survival of bacterial cells, leading to the conclusion that soil substrate plays an important role in the colonization ability.The ability of Bs MBI600 to promote plant growth and control important pathogens of cucumber or tomato was confirmed by performing a set of different experiments. Treatments with Bs MBI600 led to plants that showed increased growth, determined by measurements of several growth-related parameters (stem and root length, photosynthesis, etc.). The ability of Bs MBI600 to control tomato or cucumber pathogens was confirmed by both in vitro and in planta experiments. Bs MBI600 reduced the incidence and severity of diseases caused by: Rhizoctonia solani, Pythium ultimum, Fusarium oxysporum f.sp. radicis-lycopersici -Forl, F.oxysporum f.sp. radicis cucumerinum- Forc, P. aphanidernatum and Botrytis cinerea. Some of the mechanisms that play a role in growth promotion and disease resistance were studied and identified by transcriptomic analyses in cucumber and tomato plants that were treated with Bs MBI600. Gene expression analysis showed the activation of genes related to plant defense against phytopathogenic microorganisms (PR-proteins, POD, GluA). In addition, overexpression of genes associated with the production of substances such as ethylene, jasmonate and kinases, indicated the activation of signals that play a role in the induction of systemic resistance. Similarly, overexpression of genes associated with the production of phytohormones was observed, leading to the conclusion that treatment with Bs MBI600 activates endogenous phytohormone production.In the last chapter of this thesis, the interactions between Bs MBI600 and the phytopathogenic fungus B. cinerea were studied. The antimicrobial compounds produced by Bs MB600 contributed to the direct mode of action against the pathogen. From the transcriptomic analysis in B. cinerea after its exposure to these antimicrobial compounds, mechanisms related to the detoxification of xenobiotic substances (ABC, MFS transporters) were identified. Deletion of the mfs1 gene in B. cinerea strains showed that the functions of the fungus are affected, as well as its control by the biological control agent.Η χρήση ωφέλιμων μικροοργανισμών στην σύγχρονη γεωργία δεν αποτελεί πλέον εξαίρεση αλλά είναι απαραίτητη σε όλες τις καλλιεργητικές στρατηγικές. Τα ωφέλιμα ριζοβακτήρια αποτελούν ένα σημαντικό μέρος των εμπορικών σκευασμάτων, εξαιτίας της ικανότητας τους να προωθούν την ανάπτυξη των φυτών και να είναι αποτελεσματικά έναντι παθογόνων μικροοργανισμών. Βακτήρια που ανήκουν στο γένος Bacillus, αποτελούν την πλειοψηφία των μικροοργανισμών που διατίθενται εμπορικά ως παράγοντες βιολογικής καταπολέμησης ασθενειών των φυτών. Το εμπορικό σκεύασμα Serifel® , εμπεριέχει τον βιολογικό παράγοντα BacillussubtilisMBI600 (BsMBI600), και στην παρούσα διατριβή παρουσιάζεται μια συστηματική καταγραφή των μηχανισμών δράσης του συγκεκριμένου ωφέλιμου μικροοργανισμού. Για την καλύτερη κατανόηση των μηχανισμών δράσης του BsMBI600, πραγματοποιήθηκε αλληλούχιση του γονιδιώματος του μικροοργανισμού με Illumina HiSeq X. Τα αποτελέσματα της ανάλυσης ανέδειξαν γονίδια που σχετίζονται με την παραγωγή ουσιών που δρουν ως σήματα επικοινωνίας με τα φυτά-ξενιστές, οδηγώντας σε επιτυχημένο αποικισμό των ριζών. Επιπλέον, ταυτοποιήθηκαν γονίδια που σχετίζονται με την ικανότητα των βιολογικών παραγόντων να αυξάνουν την διαθεσιμότητα των θρεπτικών στοιχείων των φυτών, αλλά και την παραγωγή φυτοορμονών που οδηγούν στην προαγωγή της ανάπτυξης των φυτών. Τέλος, ταυτοποιήθηκαν γονίδια που σχετίζονται με την παραγωγή αντιμικροβιακών ουσιών, στο γονιδίωμα το BsMBI600.H ικανότητα αποικισμού του BsMBI600 σε ρίζες φυτών τομάτας και αγγουριάς, μελετήθηκε σε διαφορετικά υποστρώματα ανάπτυξης. Βακτηριακά κύτταρά του στελέχους, μετασχηματίσθηκαν με την εισαγωγή μια κίτρινης φθορίζουσας πρωτεΐνης, με τον τρόπο της φυσικής ιδιότητας των βακτηρίων να επιτρέπουν την εισαγωγή ξένων μορίων μέσα στο κύτταρο τους (naturaltransformation). Τα μετασχηματισμένα στελέχη του BsMBI600, μελετήθηκαν με την χρήση ανεστραμμένου φθορίζοντος μικροσκοπίου, και καταγράφηκε ο επιφυτικός αποικισμός 48 ώρες μετά την εφαρμογή. Εν συνεχεία, καταγράφθηκε η ικανότητα αποικισμού σε διαφορετικά υποστρώματα ανάπτυξης (κύβοι υδροπονίας, 2 τύποι εδαφών και αποστειρωμένο σύστημα). Ο βιολογικός παράγοντας BsMBI600, κατάφερε να αποικίσει το ριζικό σύστημα των 2 ξενιστών σε όλα τα υποστρώματα, με μεγάλες διακυμάνσεις στην διάρκεια και στην επιβίωση βακτηριακών κυττάρων, οδηγώντας στο συμπέρασμα, πως το εδαφικό υπόστρωμα παίζει σημαντικό ρόλο στην ικανότητα αποικισμού.Η ικανότητα του BsMBI 600 να προάγει την ανάπτυξη των φυτών αλλά και να ελέγχει σημαντικά παθογόνα των 2 ξενιστών, επιβεβαιώθηκε από ένα σύνολο πειραμάτων που πραγματοποιήθηκαν. Εφαρμογές με τον βιολογικό παράγοντα, οδήγησαν σε φυτά που παρουσίασαν αύξηση σε διάφορους παράγοντες (μήκος στελέχους και ρίζας, φωτοσύνθεση κ.α.). Η βιολογική δράση του έναντι φυτοπαθογόνων μικροοργανισμών επιβεβαιώθηκε τόσο με invitro πειράματα, όσο και με πειράματα σε φυτοδοχεία. Ο BsMBI600 μείωσε την εμφάνιση και την ένταση σημαντικών ασθενειών που προκαλούνται από τα παθογόνα Rhizoctonia solani, Pythium ultimum, P. Aphanidernatum,Fusarium oxysporum f.sp. radicis-lycopersici -Forl, F. oxysporum f.sp. radicis cucumerinum- Forc, και Botrytiscinerea. Μέρος των μηχανισμών που εμπλέκονται στην επαγωγή της ανάπτυξης και της άμυνας των φυτών μελετήθηκαν με ανάλυση της έκφρασης γονιδίων σε φυτά αγγουριάς και τομάτας που δέχθηκαν εφαρμογή με τον BsMBI600. Οι μετρήσεις της γονιδιακής έκφρασης έδειξαν την ενεργοποίηση γονιδίων που σχετίζονται με την άμυνα του φυτού έναντι φυτοπαθογόνων μικροοργανισμών (PR-proteins, POD, GluA). Επίσης καταγράφηκε υπερέκφραση γονιδίων που σχετίζονται με παραγωγή ουσιών όπως αιθυλένιο, ιασμονικο οξύ και κινάσες, που είναι γνωστό ότι λειτουργούν ως σήματα της επαγωγής διασυστηματικής αντοχής. Τέλος, καταγράφηκε υπερέκφραση γονιδίων που σχετίζονται με την παραγωγή φυτοορμονών, οδηγώντας στο συμπέρασμα πως η εφαρμογή με τον βιολογικό παράγοντα συμβάλει στην επαγωγή της ανάπτυξης των φυτών τομάτας και αγγουριάς μέσω της ενεργοποίησης παραγωγής ενδογενών φυτοορμονών. Τέλος μελετήθηκε η αλληλεπίδραση μεταξύ του βιολογικού παράγοντα και του φυτοπαθογόνου μύκητα B. cinerea. Οι αντιμικροβιακές ουσίες που παράγονται από τον BsMB600 συμβάλλουν στον άμεσο τρόπο δράσης του έναντι του παθογόνου. Μέσω μεταγραφωμικής ανάλυσης σε στέλεχος του B. cinerea μετά την έκθεση του στις παραπάνω ουσίες, ταυτοποιήθηκαν μηχανισμοί που σχετίζονται με την αποτοξικοποίηση ξενοβιοτικών ουσιών (ABC, MFStransporters). H διαγραφή του γονιδίου mfs1 σε στελέχη τουμύκητα, έδειξε πως επηρεάζονται ορισμένες λειτουργίες του, αλλά και η αντιμετώπιση του από τον βιολογικό παράγοντα

Detection of sdhB gene mutations in SDHI-resistant isolates of Botrytis cinerea using high resolution melting (HRM) analysis

Botrytis cinerea, is a high-risk pathogen for fungicide resistance development. Pathogen` resistance to SDHIs is associated with several mutations in sdh gene. The diversity of mutations and their differential effect on cross-resistance patterns among SDHIs and the fitness of resistant strains necessitate the availability of a tool for their rapid identification. This study was initiated to develop and validate a high-resolution melting (HRM) analysis for the identification of P225H/F/L//T, N230I and H272L/R/Y mutations. Based on the sequence of sdhB subunit of resistant and sensitive isolates, a universal primer pair was designed. The specificity of the HRM analysis primers was verified to ensure against the cross-reaction with other fungal species and its sensitivity was evaluated using concentrations of known amounts of mutant`s DNA. The melting curve analysis generated nine distinct curve profiles, enabling the discrimination of all the 4 mutations located at codon 225, the N230I mutation, the 3 mutations located in codon 272 and the non mutated isolates (isolates of wild type sensitivity). Similar results were obtained when DNA was extracted directly from artificially inoculated strawberry fruit. The method was validated by monitoring the presence of sdhB mutations in samples of naturally infected strawberry fruits and stone fruit rootstock seedling plants showing damping off symptoms. HRM analysis data were compared with a standard PIRA-PCR technique and an absolute agreement was observed suggesting that in both populations the H272R mutation was the predominant one, while H272Y, N230I and P225H were detected in lower frequencies. The results of the study suggest that HRM analysis can be a useful tool for sensate, accurate and rapid identification of several sdhB mutations in B. cinerea and it is expected to contribute in routine fungicide resistance monitoring or assessments of the effectiveness of antiresistance strategies implemented in crops heavily treated with botryticides

Fusarium equiseti as an Emerging Foliar Pathogen of Lettuce in Greece: Identification and Development of a Real-Time PCR for Quantification of Inoculum in Soil Samples

Lettuce is the most commonly cultivated leafy vegetable in Greece, available in the market throughout the year. In this study, an emerging foliar disease observed in commercial farms has been associated to the pathogen Fusarium equiseti, a member of the Fusarium incarnatum-equiseti species complex (FIESC). Thirty F. equiseti isolates obtained from symptomatic lettuce plants were identified on the basis of morphology and evaluated for their pathogenicity. The isolates were further characterized using amplification and sequence analysis of the internal transcribed region (ITS-rDNA), and of the translation elongation factor 1-alpha (TEF1-a), calmodulin (CAM), beta-tubulin (Bt), and small subunit (SSU) genes. Moreover, a novel RT-qPCR assay was developed, designing a primer pair and a probe based on the TEF1-a sequences. This assay showed high specificity, amplifying F. equiseti DNA samples, while no amplification product was observed from samples of other common soilborne fungi. The generated RT-qPCR assay could be a useful tool for the detection and quantification of F. equiseti in soil samples deriving from fields cultivated with lettuce and other leafy vegetables, hosts of this specific pathogen

Laminarin Induces Defense Responses and Efficiently Controls Olive Leaf Spot Disease in Olive

Olive leaf spot (OLS) caused by Fusicladiumoleagineum is mainly controlled using copper fungicides. However, the replacement of copper-based products with eco-friendly alternatives is a priority. The use of plant resistance-inducers (PRIs) or biological control agents (BCAs) could contribute in this direction. In this study we investigated the potential use of three PRIs (laminarin, acibenzolar-S-methyl, harpin) and a BCA (Bacillus amyloliquefaciens FZB24) for the management of OLS. The tested products provided control efficacy higher than 68%. In most cases, dual applications provided higher (p &lt; 0.05) control efficacies compared to that achieved by single applications. The highest control efficacy of 100% was achieved by laminarin. Expression analysis of the selected genes by RT-qPCR revealed different kinetics of induction. In laminarin-treated plants, for most of the tested genes a higher induction rate (p &lt; 0.05) was observed at 3 days post application. Pal, Lox, Cuao and Mpol were the genes with the higher inductions in laminarin-treated and artificially inoculated plants. The results of this study are expected to contribute towards a better understanding of PRIs in olive culture and the optimization of OLS control, while they provide evidence for potential contributions in the reduction of copper accumulation in the environment

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