Plant surfaces as vehicles of Bacillus cereus responsible of human food poisoning

• Introduction A major concern in food safety is the contamination of fresh and stored food with spoiling bacteria that provoke human poisoning. Bacillus cereus is a common food-borne pathogen responsible of important poisoning outbreaks and severe bacteraemia and septicaemia. Poisoning caused by B. cereus is classified in two main categories: emetic and diarrheic. The emetic poisoning is correlated to the production of cereulide. This toxin is very heat stable, and it can be produced in the food contaminated by B. cereus cells. Diarrheic poisoning is provoked by the enterotoxin hemolysin BL, the non-hemolytic enterotoxin and the cytotoxin K. • Objective To study the interaction of B. cereus with plants as a bacteria reservoir, and in ready-to-eat fruits and vegetables. • Materials & Methods A collection of strains implicated in food-borne outbreaks were tested in vitro for a battery of phenotypes related to bacterial multicellular behaviour and thus interaction with host. 1. Solid or liquid media were used to study biofilm formation, motility or adhesion to surfaces. 2. Leaves, fruits and vegetables (melon leaf, cucumber leaf and fruit and endive) were used to study the persistence of B. cereus over time and their distribution and organization by electron microscopy. • Results All the strains behaved similarly in vitro, only some persisted on plant surfaces. Among them, the emetic strain AH187 was selected because bacterial cells persisted on a concentration of 104-105 CFU per gram of leaf, vegetable or fruit, with a sporulation rate of 40%. The electron microscopy images showed the organization of bacteria in well-developed biofilms with visible extracellular matrix. Finally, mass spectrometry analysis proved the presence of some isoforms of cereluide on the different surfaces. • Conclusion The fact that cells of B. cereus persist in leaf surface mainly as vegetative cells are indicative of their ability to adapt to the physico-chemical changeable phyllosphere, and thus to produce the emetic toxin cereulide. The presence of spores, and the formation of biofilms can be indicative of the versatile adhesive properties of this strain to diverse surfaces. Altogether are supportive of the importance of plant surfaces either as reservoir of bacterial cells or as vehicles for further contamination and food poisoning.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Microbial profiling of a suppressiveness-induced agricultural soil amended with composted almond shells lead to isolation of new biocontrol agents

Vida et al., 2016. Microbial profiling of a suppressiveness-induced agricultural soil amended with composted almond shells lead to isolation of new biocontrol agents. Biological and integrated control of plant pathogens IOBC-WPRS Bulletin Vol. 117, 2016 pp. 140-143This study focused on the microbial profile present in an agricultural soil that becomes suppressive after the application of composted almond shells (AS) as organic amendments. The role of microbes in the suppression of Rosellinia necatrix, the causative agent of avocado white root rot, was determined after heat-treatment and complementation experiments with different types of soil. Bacterial and fungal profiles based on the 16S rRNA gene and ITS sequencing, the soil under the influence of composted almond shells revealed an increase in Proteobacteria and Ascomycota groups, as well as a reduction in Acidobacteria and Xylariales (where R. necatrix is allocated). Complementary to these findings, functional analysis by GeoChip 4.6 confirmed the improvement of a group of specific probes included in the “soil benefit” category was present only in AS-amended soils, corresponding to specific microorganisms previously described as potential biocontrol agents, such as Pseudomonas spp., Burkholderia spp. or Actinobacteria. Based in such data, a model for the microbial-based suppressiveness is proposed and further isolation of representative microorganisms were performed.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

De novo analysis of the haustorial transcriptome of the cucurbit powdery mildew fungus Podosphaera xanthii reveals new candidate secreted effector proteins

Backgrounds. Cucurbit crops are affected, among other pathogens, by the obligate biotrophic fungus Podosphaera xanthii, the main causal agent of powdery mildew in cucurbits. This fungus develops a specialized structure of parasitism termed haustorium. Haustoria are developed into epidermal cells and are responsible for nutrients uptake and effectors delivery. Objectives. The aim of this study was to obtain the haustorial transcriptome of P. xanthii to complete the panel of effector candidates of this fungal pathogen. Methods. To obtain the haustorial transcriptome, we have developed an effective method for isolation of haustoria without contaminants by flow cytometry. The cDNA library was built using a combination of dT primers and random primers followed by a depletion of ribosomal sequences. Sequencing was carried out by Illumina NextSeq550. Conclusions. After bioinformatic analysis, we were able to identify 25 new effector candidates secreted by the classic pathway (with signal peptide) and 269 new candidates secreted by the non-classic pathway (without signal peptide). Most proteins had no functional annotation. By protein modelling and ligand predictions, we are now being able to assign putative functions to some of these candidates to select those with potential roles in pathogenesis for subsequent functional in vivo analysis by HIGS (host-induced gene silencing). By these approaches, we are starting to shed some light into the molecular mechanisms of pathogenesis in this very important pathogen of cucurbits.This work was supported by a grant from the Ministerio de Economía y Competitividad (AGL2013-41938-R), co-financed with FEDER funds (EU). A grant form Universidad de Málaga, Campus de Excelencia Internacional Andalucía Tech, is also acknowledged

Resistance to SDHI fungicides in Botrytis cinerea from strawberry fields in Spain

Gray mold, caused by Botrytis cinerea Pers., is one of the most economically important diseases of strawberries and its control involves the application of fungicides throughout the strawberry growing season. Succinate dehydrogenase inhibitors (SDHIs) constitute a novel class of fungicides representing new alternatives for strawberry growers. In the present study, B. cinerea isolates were used to determine the effective concentration that reduces mycelial growth by 50% (EC50) and to obtain discriminatory doses to monitor SDHI fungicides over the course of three-year monitoring period. The overall frequencies of resistance to the SDHI fungicides boscalid, fluopyram, fluxapyroxad and penthiopyrad were 56.9, 6.9, 12.9, and 24.6%, respectively. Four SDHI resistance patterns were observed in our population. Patterns I (resistance to boscalid) and II (resistance to boscalid and penthiopyrad) were associated with the amino acid substitutions H272R/Y; pattern III (resistance to boscalid, fluxapyroxad, and penthiopyrad) was associated only with the H272Y mutation; and finally, pattern IV (resistance to boscalid, fluopyram, fluxapyroxad and penthiopyrad) was associated with the N230I allele in the SdhB subunit. For gray mold management, it is suggested that the simultaneous use of boscalid and penthiopyrad should be limited to one application per season. The use of fluxapyroxad and fluopyram could be used as valid SDHI alternatives for our strawberry growers, but they should be applied with caution.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Deciphering the suppressive soil microbiota from an avocado crop

Comunicación en pósterDifferent strategies based on ecological principles have been approached in sustainable agriculture causing positive effects, including the induction of soil suppressiveness against a wide range of soilborne pathogens. Suppressiveness against the phytopathogenic fungus Rosellinia necatrix was observed after the application of composted almond shells in avocado crops. Previous works have analyzed the use of this traditional strategy and applied new microbial community analysis techniques in order to help in the identification of targeted sustainable agricultural strategies. These studies have focused on the microbial profile from an induced-suppressive soil where the soil microbiome had a proven essential role. Microbial profiles based on the 16S rRNA gene and ITS regions sequencing were analysed and an increase in Gammaproteobacteria and Dothideomycetes groups, as well as a reduction in Xylariales (where R. necatrix is allocated) were observed. These results led to the bacterial isolation of different groups of Gammaproteobacteria from this suppressive soil in order to identify new strains with biological control properties. Different characterization tests were performed, and a final selection of representative strains belonging to the genus Pseudomonas and related groups showed, all of them, plant disease protection abilities. Moreover, using previously described biological control agents against R. necatrix, a bacterial synthetic community have been design in order to improve the knowledge of the multitrophic interactions that occur during biological control process.This work was supported by Plan Nacional I+D+I (MINECO, Spain) (AGL2014-52518-C2-IR) and co-financed by FEDER funds (EU). C.Vida was supported by a PhD fellowship from the FPI program of MINECO. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Isolation, characterization and selection of Pseudomonas spp. as biological control agents from a suppressive soil

Bacterial profile from a suppressive soil against Rosellinia necatrix, fungal pathogen of avocado roots, were obtained by 16S rRNA gene sequencing. The results revealed a significant increase in the bacterial class of Gammaproteobacteria, especially in some antagonistic representatives of Pseudomonas spp. For this reason, a collection of 246 bacterial isolates was obtained from this suppressive soil in order to identify new strains with antifungal activity against this fungal phytopathogen. First, we performed an isolation on a selective medium for Pseudomonas-like microorganisms. Then, we used different characterization tests in order to analyse the bacterial collection, including the identification of the general metabolic profile of glucose, the profiling of antifungals produced, both the putative production of antifungal compounds and lytic exoenzymes, as well as the evaluation of traits related with beneficial effects on plants. A final selection of representative strains resulted in antifungal isolates belonging to the genus Pseudomonas and related groups. These selected strains were tested for plant protection by an in vivo experiment using avocado and wheat plants challenged by the pathogen R. necatrix, showing all of them an antifungal ability and plant disease protection.This work was supported by Plan Nacional I+D+I from MINECO (Spain) (AGL2014-52518-C2-IR) and co-financed by FEDER funds (EU). C.Vida was supported by a PhD fellowship from the FPI program of MINECO.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Extracellular matrix components are required to protect bacillus subtilis from pseudomonas invasion and co-colonization of plants

Plants are colonized by a vast variety of microbes. Among them, bacteria are the most predominant and are able to adapt to environmental changes and interact with other microorganisms using a wide array of molecules, metabolic plasticity and secretion systems. One way bacteria have evolved to succeed in this competitive scenario is the formation of biofilms which provides protection to the cells, modulates the flux of signals and controls cellular differentiation. Thus, efforts are encouraged to really determine the functionality of the bacterial extracellular matrix. In this study, we have employed microbiological and microscopic techniques to study the interaction between Bacillus subtilis 3610 and Pseudomonas chlororaphis PCL1606. We demonstrate the important role of the extracellular matrix in protecting B. subtilis colonies from infiltration by Pseudomonas. Furthermore, time-lapse confocal laser scanning microscopy (CLSM) analyses of the bacterial interactions have permitted to complete the study of the bacterial behaviors and to measure bacterial expansion rates. Surprising, we find that the Pseudomonas type VI secretion system (T6SS) is required in the cell-to-cell contact with matrix-impaired B. subtilis cells, revealing a novel role for T6SS against Gram-positive bacteria. In response to P. chlororaphis infiltration, we find that B. subtilis activates sporulation and expresses motility-related genes. Confocal microscopy of the bacterial interactions using plant organs highlights the functional importance of these different bacterial strategies in their coexistence as stable bacterial communities. The findings further our understanding of the functional role played by biofilms in mediating bacterial social interactions.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Analysis of the molecular machinery implicated in multicellularity in bacillus cereus

Introduction: Bacillus cereus is a Gram-positive bacterium usually implicated in food poisoning outbreaks and human infections that sometimes result fatal. These events are closely related to the assembly of a biofilm that serves as a reservoir of cells, a nest for sporulation and protection from environmental stresses, host defenses or chemotherapy. Objectives: To perform a comprehensive comparative study of biofilm and planktonic cells to: i) delineate the molecular machinery implicated in the different steps of the biofilm life cycle, and ii) define new genes dedicated to biofilm formation. Materials & methods: Bacteria were grown under biofilm inducing conditions. Biofilm cells were separated from planktonic cells at different times and their whole mRNA was isolated, sequenced and analyzed. Results: Our results reveal a high number of genes associated to biofilm, many of them with unknown function, but highly conserved in others bacterial species. Besides, we found global changes in cell wall synthesis, metabolism and interspecies interaction molecules. ​ Conclusions: The interaction of B. cereus with other bacteria is conditioned by secondary metabolites, which are apparently overexpressed in biofilm. On the other hand, toxins are mainly expressed in planktonic cells, which are more oriented to interact with its hosts. These results reveal the defense and attacking positions of B. cereus in biofilm vs planktonic states.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Criticality analysis for improving maintenance, felling and pruning cycles in power lines

16th IFAC Symposium on Information Control Problems in Manufacturing INCOM 2018 Bergamo, Italy, 11–13 June 2018. Edited by Marco Macchi, László Monostori, Roberto PintoThis paper deals with the process of criticality analysis in overhead power lines, as a tool to improve maintenance, felling & pruning programs. Felling & pruning activities are tasks that utility companies must accomplish to respect the servitudes of the overhead lines, concerned with distances to vegetation, buildings, infrastructures and other networks crossings. Conceptually, these power lines servitudes can be considered as failure modes of the maintainable items under our analysis (power line spans), and the criticality analysis methodology developed, will therefore help to optimize actions to avoid these as other failure modes of the line maintainable items. The approach is interesting, but another relevant contribution of the paper is the process followed for the automation of the analysis. Automation is possible by utilizing existing companies IT systems and databases. The paper explains how to use data located in Enterprise Assets Management Systems, GIS and Dispatching systems for a fast, reliable, objective and dynamic criticality analysis. Promising results are included and also discussions about how this technique may result in important implications for this type of businesse

Resistance to the SDHI fungicides boscalid and fluopyram in Podosphaera xanthii from commercial cucurbit fields in Spain

Powdery mildew elicited by Podosphaera xanthii is a devastating disease of cucurbits worldwide and one of the most important diseases affecting these crops in Spain. Application of fungicides is the main control practice for managing P. xanthii; however, isolates resistant to multiple classes of site-specific fungicides have been recently reported in the Spanish cucurbit powdery mildew population. Succinate dehydrogenase inhibitors (SDHIs) constitute a relatively novel class of fungicides registered for powdery mildew control representing new alternatives for cucurbit growers. In the present study, 30 P. xanthii isolates were used to determine the effective concentration that reduces mycelial growth by 50% (EC50) to boscalid and fluopyram. The present study was also conducted to obtain discriminatory doses to monitor SDHI fungicide resistance in 180 P. xanthii isolates collected from several commercial cucurbit fields in Spain during 2017-2018. Three SDHI resistance patterns were observed in our population, which include patterns I (resistance to boscalid), II (resistance to fluopyram), and III (resistance to boscalid and fluopyram). The amino acid changes associated with these resistance patterns in the Sdh protein were also examined. Based on our results, SDHI fungicides are good alternatives for cucurbit powdery mildew control, although they should be applied with caution.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tec