Toxin Induction and Protein Extraction from Fusariumspp. Cultures for Proteomic Studies

Fusaria are filamentous fungi able to produce different toxins. Fusarium mycotoxins such as deoxynivalenol, nivalenol, T2, zearelenone, fusaric acid, moniliformin, etc... have adverse effects on both human and animal health and some are considered as pathogenicity factors. Proteomic studies showed to be effective for deciphering toxin production mechanisms (Taylor et al., 2008) as well as for identifying potential pathogenic factors (Paper et al., 2007, Houterman et al., 2007) in Fusaria. It becomes therefore fundamental to establish reliable methods for comparing between proteomic studies in order to rely on true differences found in protein expression among experiments, strains and laboratories. The procedure that will be described should contribute to an increased level of standardization of proteomic procedures by two ways. The filmed protocol is used to increase the level of details that can be described precisely. Moreover, the availability of standardized procedures to process biological replicates should guarantee a higher robustness of data, taking into account also the human factor within the technical reproducibility of the extraction procedure

A European Database of Fusarium graminearum and F-culmorum Trichothecene Genotypes

Fusarium species, particularly Fusarium graminearum and F culmorum, are the main cause of trichothecene type B contamination in cereals. Data on the distribution of Fusarium trichothecene genotypes in cereals in Europe are scattered in time and space. Furthermore, a common core set of related variables (sampling method, host cultivar, previous crop, etc.) that would allow more effective analysis of factors influencing the spatial and temporal population distribution, is lacking. Consequently, based on the available data, it is difficult to identify factors influencing chemotype distribution and spread at the European level. Here we describe the results of a collaborative integrated work which aims (1) to characterize the trichothecene genotypes of strains from three Fusarium species, collected over the period 2000-2013 and (2) to enhance the standardization of epidemiological data collection. Information on host plant, country of origin, sampling location, year of sampling and previous crop of 1147 F graminearurn, 479 F culmorum, and 3 F cortaderiae strains obtained from 17 European countries was compiled and a map of trichothecene type B genotype distribution was plotted for each species. All information on the strains was collected in a freely accessible and updatable database (www.catalogueeu.luxmcc.lu), which will serve as a starting point for epidemiological analysis of potential spatial and temporal trichothecene genotype shifts in Europe. The analysis of the currently available European dataset showed that in F. grarninearum, the predominant genotype was 15-acetyldeoxynivalenol (15-ADON) (82.9%), followed by 3-acetyldeoxynivalenol (3-ADON) (13.6%), and nivalenol (NIV) (3.5%). In F culmorum, the prevalent genotype was 3-ADON (59.9%), while the NIV genotype accounted for the remaining 40.1%. Both, geographical and temporal patterns of trichothecene genotypes distribution were identified.Ministere de l'Agriculture, de la Viticulture et de la Protection des Consommateurs-Administration des Services Techniques de l'Agriculture; M.I.U.R. Project AGROGEN (Laboratory of GENomics for traits of AGROnomic importance in durum wheat: Identification of useful genes, functional analysis and assisted selection by biological markers for the development of the national seed chain) [602/Ric]; Felix Thornley Cobbold Trust; John Oldacre Foundation; Ministry of Agriculture of the Czech RepublicMinistry of Agriculture, Czech Republic [800415]; Spanish Ministry MINECOSpanish Government [AGL201.4-53928-C2-2-R]; Ministry of Agriculture and Food, Norway; Federal Ministry of Education and Research (BMBF) (GABI-KANADA), BonnFederal Ministry of Education & Research (BMBF) [FKZ 0313711A]; German Academic Exchange Service (DAAD), BonnDeutscher Akademischer Austausch Dienst (DAAD) [A/06/92183]; Finnish Ministry of Agriculture and Forestry; Direction Generale de l'Agriculture, Direction de la Recherche [D31-3159, D31-1162, D31-7055]; P.O.R. SARDEGNA F.S.; Danish Directorate for Food, Fisheries and Agri Business [FFS05-3]; Academy of FinlandAcademy of Finland [126917, 131957, 250904, 252162, 267188, 266984]; Olvi Foundation; Turku University Foundation; CIMO travel grant; Nordic network project New Emerging Mycotoxins and Secondary Metabolites in Toxigenic Fungi of Northern Europe - Nordic Research Board [090014]The Luxembourg institute of Science and Technology, LU, acknowledges the Ministere de l'Agriculture, de la Viticulture et de la Protection des Consommateurs-Administration des Services Techniques de l'Agriculture for financially supporting the Sentinelle project. The work on Italian strains has been financially supported through the M.I.U.R. Project AGROGEN (Laboratory of GENomics for traits of AGROnomic importance in durum wheat: Identification of useful genes, functional analysis and assisted selection by biological markers for the development of the national seed chain) (D. D. 14.03.2005 n. 602/Ric). Funding for the research of Ryan Basler was provided by Felix Thornley Cobbold Trust and the John Oldacre Foundation.; The work of JC was supported by the Ministry of Agriculture of the Czech Republic, Project No. 800415. The research of MG and PG was supported by the Spanish Ministry MINECO (AGL201.4-53928-C2-2-R). The Ministry of Agriculture and Food, Norway funded the work of IH. The research of TM was funded by the Federal Ministry of Education and Research (BMBF) (GABI-KANADA #FKZ 0313711A), Bonn and by the German Academic Exchange Service (DAAD), Bonn (code no.: A/06/92183). PP acknowledges the Finnish Ministry of Agriculture and Forestry for funding the project FinMyco on Fusarium and mycotoxins in Finland. The research of JS was funded by the Direction Generale de l'Agriculture, Direction de la Recherche (ref. D31-3159, D31-1162, D31-7055), in the framework of a project entitled Caracterization et dynamique des fusarioses sur mais en Region Wallonne. BS acknowledges support by P.O.R. SARDEGNA F.S.E. 2007-2013-Obiettivo competitivita regionale e occupazione, Asse IV Capitale umano, Linea di Attivita 1.3.1 (research project Identification of natural and natural-like molecules inhibiting mycotoxin biosynthesis by Fusaria pathogenic on cereals). UT thanks the Danish Directorate for Food, Fisheries and Agri Business grant FFS05-3 for financial support. The work of TY was financially supported by the Academy of Finland (no. 126917, 131957, 250904, 252162, 267188, and 266984), Olvi Foundation, Turku University Foundation, a CIMO travel grant to Taha Hussien, and the Nordic network project New Emerging Mycotoxins and Secondary Metabolites in Toxigenic Fungi of Northern Europe (project 090014), which was funded by the Nordic Research Board

Validation of a Quick PCR Method Suitable for Direct Sequencing: Identification of Fusarium Fungal Species and Chemotypes for Preventive Approaches in Food Safety

Za razvoj preventivnih mjera koje se provode radi poboljšanja sigurnosti hrane neophodno je odrediti toksičnost plijesni roda Fusarium, pa se sekvenciranjem i genetskom kemotipizacijom pomoću metode lančane reakcije polimerazom određuju vrste ovoga roda plijesni. U radu je predložen brzi protokol za standardni postupak utvrđivanja vrste plijesni sekvenciranjem faktora elongacije 1α i multipleks kemotipizacijom s pomoću gena Tri12. Provedena je i statistička obrada dobivenih podataka. Kao podloga za uzgoj plijesni upotrijebljen je filter papir Miracloth, a DNA je iz plijesni izdvojena pomoću mikrovalova. Ispitano je 75 sojeva plijesni Fusarium culmorum i Fusarium graminearum, te je zaključeno da se ovim postupkom mogu uspješno odrediti vrste roda Fusarium.Species determination by sequencing and PCR genetic chemotyping, used to determine the toxigenic potential of Fusarium strains, is fundamental for developing preventive strategies in food safety. Here we propose and statistically validate a quick protocol for standardizing the procedure of species determination by sequencing of the elongation factor 1-α and multiplex genetic chemotyping using the Tri12 gene, based on fungal growth on Miracloth tissue coupled with microwave extraction. The test was validated on 75 Fusarium culmorum and Fusarium graminearum strains

Evaluation of in-vitro methods to select effective streptomycetes against toxigenic fusaria

Biocontrol microorganisms are emerging as an effective alternative to pesticides. Ideally, biocontrol agents (BCAs) for the control of fungal plant pathogens should be selected by an in vitro method that is high-throughput and is predictive of in planta efficacy, possibly considering environmental factors, and the natural diversity of the pathogen. The purpose of our study was (1) to assess the effects of Fusarium strain diversity (N = 5) and culture media (N = 6) on the identification of biological control activity of Streptomyces strains (N = 20) against Fusarium pathogens of wheat in vitro and (2) to verify the ability of our in vitro screening methods to simulate the activity in planta. Our results indicate that culture media, Fusarium strain diversity, and their interactions affect the results of an in vitro selection by dual culture assay. The results obtained on the wheat-based culture media resulted in the highest correlation score (r = 0.5) with the in planta root rot (RR) inhibition, suggesting that this in vitro method was the best predictor of in planta performance of streptomycetes against Fusarium RR of wheat assessed as extension of the necrosis on the root. Contrarily, none of the in vitro plate assays using the media tested could appropriately predict the activity of the streptomycetes against Fusarium foot rot symptoms estimated as the necrosis at the crown level. Considering overall data of correlation, the activity in planta cannot be effectively predicted by dual culture plate studies, therefore improved in vitro methods are needed to better mimic the activity of biocontrol strains in natural conditions. This work contributes to setting up laboratory standards for preliminary screening assays of Streptomyces BCAs against fungal pathogens

Genetic variability, chemotype distribution, and aggressiveness of Fusarium culmorum on durum wheat in Tunisia

Fusarium culmorum is the most commonly reported root rot pathogen in Tunisian durum wheat. Isolates of the pathogen from four durum wheat growing areas in the north of Tunisia were analyzed for their chemotypes. Two chemotypes were detected at unequal abundance (96% of 3-ADON and 4% of NIV). Distribution of a SNP mutation located at the position 34 bp after the first exon of the EF-1α partial sequence was analysed, to verify whether the haplotype was specifically associated to Fusarium root rot. A and T haplotypes were homogeneously distributed in three different Tunisian regions (Mateur, Beja and Bousalem) but not for the region of Bizerte, from which greatest number of A haplotype strains were detected. The isolates were tested for their virulence under glasshouse conditions, and a mean of 91% of crown and root infection was observed. Chemotype influenced virulence, but there was no significant influence of the geographical origin or haplotype on virulence. The distribution of three inter simple sequence repeats (ISSR) was examined, to better understand the structure of F. culmorum populations in Tunisia. A total of 27 fragments were obtained with eight polymorphic bands. Cluster analysis showed a high level of similarity between isolates. Analysis of molecular variance confirmed that there was little genetic differentiation among F. culmorum strains from different locations

Global Geographic Distribution and Host Range of Fusarium circinatum, the Causal Agent of Pine Pitch Canker

Funding: This study was financially supported by COST Action FP1406 (PINESTRENGTH), the Estonian Science Foundation grant PSG136, the Forestry Commission, United Kingdom, the Phytophthora Research Centre Reg. No. CZ.02.1.01/0.0/0.0/15_003/0000453, a project co-financed by the European Regional Development Fund. ANSES is supported by a grant managed by the French National Research Agency (ANR) as part of the “Investissements d’Avenir” programme (ANR-11-LABX-0002-01, Laboratory of ExcellenceARBRE). SW was partly supported by BBSRC Grant reference BB/L012251/1 “Promoting resilience of UK tree species to novel pests & pathogens: ecological & evolutionary solutions (PROTREE)” jointly funded by BBSRC, Defra, ESRC, the Forestry Commission, NERC and the Scottish Government, under the Tree Health and Plant Biosecurity Initiative. Annual surveys in Switzerland were financially supported by the Swiss Federal Office for the Environment FOEN. Acknowledgments: Andrea Kunova and Cristina Pizzatti are acknowledged for the assistance in the sampling. Thanks are due to Dina Ribeiro and Helena Marques from ICNF-Portuguese Forest Authority for providing location coordinates. We thank three anonymous reviwers for valuable corrections and suggestions.Peer reviewedPublisher PD

A European Database of Fusarium graminearum and F. culmorum Trichothecene Genotypes

. Fusarium species, particularly Fusarium graminearum and F culmorum, are the main cause of trichothecene type B contamination in cereals. Data on the distribution of Fusarium trichothecene genotypes in cereals in Europe are scattered in time and space. Furthermore, a common core set of related variables (sampling method, host cultivar, previous crop, etc.) that would allow more effective analysis of factors influencing the spatial and temporal population distribution, is lacking. Consequently, based on the available data, it is difficult to identify factors influencing chemotype distribution and spread at the European level. Here we describe the results of a collaborative integrated work which aims (1) to characterize the trichothecene genotypes of strains from three Fusarium species, collected over the period 2000-2013 and (2) to enhance the standardization of epidemiological data collection. Information on host plant, country of origin, sampling location, year of sampling and previous crop of 1147 F graminearurn, 479 F culmorum, and 3 F cortaderiae strains obtained from 17 European countries was compiled and a map of trichothecene type B genotype distribution was plotted for each species. All information on the strains was collected in a freely accessible and updatable database (www.catalogueeu.luxmcc.lu), which will serve as a starting point for epidemiological analysis of potential spatial and temporal trichothecene genotype shifts in Europe. The analysis of the currently available European dataset showed that in F. grarninearum, the predominant genotype was 15-acetyldeoxynivalenol (15-ADON) (82.9%), followed by 3-acetyldeoxynivalenol (3-ADON) (13.6%), and nivalenol (NIV) (3.5%). In F culmorum, the prevalent genotype was 3-ADON (59.9%), while the NIV genotype accounted for the remaining 40.1%. Both, geographical and temporal patterns of trichothecene genotypes distribution were identified.</p

A European Database of Fusarium graminearum and F-culmorum Trichothecene Genotypes

Fusarium species, particularly Fusarium graminearum and F culmorum, are the main cause of trichothecene type B contamination in cereals. Data on the distribution of Fusarium trichothecene genotypes in cereals in Europe are scattered in time and space. Furthermore, a common core set of related variables (sampling method, host cultivar, previous crop, etc.) that would allow more effective analysis of factors influencing the spatial and temporal population distribution, is lacking. Consequently, based on the available data, it is difficult to identify factors influencing chemotype distribution and spread at the European level. Here we describe the results of a collaborative integrated work which aims (1) to characterize the trichothecene genotypes of strains from three Fusarium species, collected over the period 2000-2013 and (2) to enhance the standardization of epidemiological data collection. Information on host plant, country of origin, sampling location, year of sampling and previous crop of 1147 F graminearurn, 479 F culmorum, and 3 F cortaderiae strains obtained from 17 European countries was compiled and a map of trichothecene type B genotype distribution was plotted for each species. All information on the strains was collected in a freely accessible and updatable database (www.catalogueeu.luxmcc.lu), which will serve as a starting point for epidemiological analysis of potential spatial and temporal trichothecene genotype shifts in Europe. The analysis of the currently available European dataset showed that in F. grarninearum, the predominant genotype was 15-acetyldeoxynivalenol (15-ADON) (82.9%), followed by 3-acetyldeoxynivalenol (3-ADON) (13.6%), and nivalenol (NIV) (3.5%). In F culmorum, the prevalent genotype was 3-ADON (59.9%), while the NIV genotype accounted for the remaining 40.1%. Both, geographical and temporal patterns of trichothecene genotypes distribution were identified