Matias Pasquali

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. graminearum, 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. graminearum, 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.

Fusarium culmorum: causal agent of foot and root rot and head blight on wheat

UNLABELLED Fusarium culmorum is a ubiquitous soil-borne fungus able to cause foot and root rot and Fusarium head blight on different small-grain cereals, in particular wheat and barley. It causes significant yield and quality losses and results in contamination of the grain with mycotoxins. This review summarizes recent research activities related to F. culmorum, including studies into its population diversity, mycotoxin biosynthesis, mechanisms of pathogenesis and resistance, the development of diagnostic tools and preliminary genome sequence surveys. We also propose potential research areas that may expand our basic understanding of the wheat-F. culmorum interaction and assist in the management of the disease caused by this pathogen. TAXONOMY Fusarium culmorum (W.G. Smith) Sacc. Kingdom Fungi; Phylum Ascomycota; Subphylum Pezizomycotina; Class Sordariomycetes; Subclass Hypocreomycetidae; Order Hypocreales; Family Nectriaceae; Genus Fusarium. DISEASE SYMPTOMS Foot and root rot (also known as Fusarium crown rot): seedling blight with death of the plant before or after emergence; brown discoloration on roots and coleoptiles of the infected seedlings; brown discoloration on subcrown internodes and on the first two/three internodes of the main stem; tiller abortion; formation of whiteheads with shrivelled white grains; Fusarium head blight: prematurely bleached spikelets or blighting of the entire head, which remains empty or contains shrunken dark kernels. IDENTIFICATION AND DETECTION: Morphological identification is based on the shape of the macroconidia formed on sporodochia on carnation leaf agar. The conidiophores are branched monophialides, short and wide. The macroconidia are relatively short and stout with an apical cell blunt or slightly papillate; the basal cell is foot-shaped or just notched. Macroconidia are thick-walled and curved, usually 3-5 septate, and mostly measuring 30-50 × 5.0-7.5 μm. Microconidia are absent. Oval to globose chlamydospores are formed, intercalary in the hyphae, solitary, in chains or in clumps; they are also formed from macroconidia. The colony grows very rapidly (1.6-2.2 cm/day) on potato dextrose agar (PDA) at the optimum temperature of 25 °C. The mycelium on PDA is floccose, whitish, light yellow or red. The pigment on the reverse plate on PDA varies from greyish-rose, carmine red or burgundy. A wide array of polymerase chain reaction (PCR) and real-time PCR tools, as well as complementary methods, which are summarised in the first two tables, have been developed for the detection and/or quantification of F. culmorum in culture and in naturally infected plant tissue. HOST RANGE Fusarium culmorum has a wide range of host plants, mainly cereals, such as wheat, barley, oats, rye, corn, sorghum and various grasses. In addition, it has been isolated from sugar beet, flax, carnation, bean, pea, asparagus, red clover, hop, leeks, Norway spruce, strawberry and potato tuber. Fusarium culmorum has also been associated with dermatitis on marram grass planters in the Netherlands, although its role as a causal agent of skin lesions appears questionable. It is also isolated as a symbiont able to confer resistance to abiotic stress, and has been proposed as a potential biocontrol agent to control the aquatic weed Hydrilla spp. USEFUL WEBSITES http://isolate.fusariumdb.org/; http://sppadbase.ipp.cnr.it/; http://www.broad.mit.edu/annotation/genome/fusarium_group/MultiHome.html; http://www.fgsc.net/Fusarium/fushome.htm; http://plantpath.psu.edu/facilities/fusarium-research-center; http://www.phi-base.org/; http://www.uniprot.org/; http://www.cabi.org/; http://www.indexfungorum.org/

Genetic Fusarium chemotyping as a useful tool for predicting nivalenol contamination in winter wheat.

Fusarium graminearum [teleomorph Gibberella zeae] and Fusarium culmorum together with Fusarium poae are the main species known to produce nivalenol (NIV). The NIV content in wheat (Triticum aestivum L.) harvested in Luxembourg was investigated in 2007 and 2008 at 17 different locations. Species determination and genetic chemotyping of F. graminearum and F. culmorum were used to understand the spatial distribution of NIV producers in wheat from Luxembourg. Three hundred thirteen F. graminearum, 175 F. culmorum and 117 F. poae strains respectively were isolated. Chemotypes of the first two species were determined by PCR and confirmed on a sub-sample of single isolates by LC-MS/MS analysis. The 15-acetylated DON chemotype of F. graminearum was dominant in both years representing 94.2% of the population while the NIV chemotype represented 5.8%. The F. culmorum chemotypes were rather evenly distributed, with 3-acetylated DON and NIV profiles present with similar abundances (53.2% and 46.8%, respectively). NIV presence in wheat flour obtained from the 17 sites was correlated with the number of F. culmorum (NIV chemotype) isolated from 100 seeds, suggesting its primary role in NIV production on grains. The predictive power for identifying NIV contamination in grains based on NIV chemotype presence was confirmed by coupling the isolation procedure with a cut-off value, resulting in the successful identification (100%, p=0.008) of NIV contamination in grains collected from 9 additional experimental sites. In conclusion, the results highlight the importance of chemotyping for improved prediction of toxin contamination in wheat.

Identification of Race 1 of Fusarium oxysporum f. sp. lactucae on Lettuce by Inter-Retrotransposon Sequence-Characterized Amplified Region Technique

ABSTRACT Fusarium wilt of lettuce, caused worldwide by Fusarium oxysporum f. sp. lactucae, is an emerging seed-transmitted disease on Lactuca sativa. In order to develop a molecular diagnostic tool for identifying race 1 (VCG0300) of the pathogen on vegetable samples, an effective technique is presented. Inter-retrotransposon amplified polymorphism polymerase chain reaction (PCR), a technique based on the amplification of genomic regions between long terminal repeats, was applied. It was shown to be useful for grouping F. oxysporum f. sp. lactucae race 1 isolates. Inter-retrotransposon sequence-characterized amplified regions (IR-SCAR) was used to develop a specific set of PCR primers to be utilized for differentiating F. oxysporum f. sp. lactucae isolates from other F. oxysporum isolates. The specific primers were able to uniquely amplify fungal genomic DNA from race 1 isolates obtained in Italy, Portugal, the United States, Japan, and Taiwan. The primers also were specific to pathogen DNA obtained from artificially infected lettuce seed and naturally and artificially infected plants.

Genetic approaches to chemotype determination in type B-trichothecene producing Fusaria.

This review summarises the genetic methods used for chemotype determination of the main Fusarium type B-trichothecene producing species. Literature on Fusarium chemotype epidemiology over the last 15 years is reviewed in order to describe temporal and spatial chemotype distribution of these fungi worldwide. Genetic approaches used for chemotype determination are also reviewed and discussed, highlighting successes and potential pitfalls of the technique. Results from both genetic and chemical approaches are summarised to compare reliability, advantages and limitations of the two methods. Potential applications of genetic chemotyping to toxigenic Fusarium species are evaluated in the light of improving food safety of agricultural products. The use of chemotype determination in population studies, toxin prediction as well as for breeding purpose is described.

Fusarium head blight and associated mycotoxin occurrence on winter wheat in Luxembourg in 2007/2008.

Fusarium head blight (FHB) is among the major causes of reduced quality in winter wheat and its products. In addition, the causal fungi produce a variety of toxins. A relatively high FHB infection rate in winter wheat was observed in 2007 and 2008 in Luxembourg. A fusariotoxin survey was carried out in 17 different geographical locations. Three groups of Fusarium mycotoxins (trichothecenes A and B and zearalenone) were analysed by a multi-detection HPLC–MS/MS method. Fusarium strains were also investigated by morphological and molecular methods. In addition, questionnaires relating to cultural practices were sent to the farmers managing the 17 fields investigated. FHB prevalence ranged from 0.3 to 65.8% (mean: 8.5%) in 2007 and from 0 to 24.5% (mean: 8.3%) in 2008. Results of morphological and molecular identification showed that the most common species isolated from diseased wheat spikes was F. graminearum (33.1%), followed by F. avenaceum (20.3%) and F. poae (17.8%). The chemical analysis revealed that 75% of the investigated fields were contaminated by deoxynivalenol (DON, range 0–8111 µg/kg). The preceding crop was highly and significantly correlated to the number of grains infected and had a significant impact on disease prevalence (p = 0.025 and 0.017, respectively, Fishers F-test). A trend was found for maize as the preceding crop (p = 0.084, Tukeys test) to predict the amount of DON in the fields. This is the first report on the occurrence of DON and ZON in naturally infected wheat grains sampled from Luxembourg.

Vegetative Compatibility Groups of Fusarium oxysporum f. sp. lactucae from Lettuce

Fusarium oxysporum f. sp. lactucae, the causal agent of Fusarium wilt of lettuce, has been reported in three continents in the last 10 years. Forty-seven isolates obtained from infected plants and seed in Italy, the United States, Japan, and Taiwan were evaluated for pathogenicity and vegetative compatibility. Chlorate-resistant, nitrate-nonutilizing mutants were used to determine genetic relatedness among isolates from different locations. Using the vegetative compatibility group (VCG) approach, all Italian and American isolates, type 2 Taiwanese isolates, and a Japanese race 1 were assigned to the major VCG 0300. Taiwanese isolates type 1 were assigned to VCG 0301. The hypothesis that propagules of Fusarium oxysporum f. sp. lactucae that caused epidemics on lettuce in 2001-02 in Italian fields might have spread via import and use of contaminated seeds is discussed.

Differences between the succinate dehydrogenase sequences of isopyrazam sensitive Zymoseptoria tritici and insensitive Fusarium graminearum strains

Forty-one Zymoseptoria tritici strains isolated in Luxembourg between 2009 and 2010 were highly sensitive towards the new succinate dehydrogenase inhibitor (SDHI) isopyrazam, with concentrations inhibiting fungal growth by 50% (EC50) ranging from 0.0281 to 4.53μM, whereas 41 Fusarium graminearum strains isolated in Europe and Northern America between 1969 and 2009 were insensitive with the average rate of inhibition converging towards 28% with increasing isopyrazam concentration. Seven isolates of both species covering the range of isopyrazam sensitivities observed in the present study were selected for the sequencing of the subunits B, C and D of the succinate dehydrogenase (sdh) gene. Predicted sdh amino acid sequences of subunits B, C and D were identical among F. graminearum strains. By comparing with fungal strains where resistance towards SDHIs was previously reported, three variations were unique to F. graminearum; B-D130N located in the iron-sulfur cluster [2Fe-2S], B-A275T located in the [3Fe-4S] cluster and an additional S at amino acid position 83-84 of sdhC, probably modifying structurally the ubiquinone binding site and therefore the biological activity of the fungicide. No variation was found among the Z. tritici strains in subunits B and D. Two variations were observed within the subunit C sequences of Z. tritici strains: C-N33T and C-N34T. The difference in EC50 values between Z. tritici strains with the NN and TT configuration was non-significant at P=0.289. Two outliers in the Z. tritici group with significantly higher EC50 values that were not related to mutations in the sdhB, sdhC, or sdhD were detected. The role of isopyrazam for the control of F. graminearum and Z. tritici in Luxembourg is discussed.

FcStuA from Fusarium culmorum controls wheat foot and root rot in a toxin dispensable manner.

Fusarium culmorum is one of the most harmful pathogens of durum wheat and is the causal agent of foot and root rot (FRR) disease. F. culmorum produces the mycotoxin deoxynivalenol (DON) that is involved in the pathogenic process. The role of the gene FcStuA, a StuA ortholog protein with an APSES domain sharing 98.5% homology to the FgStuA protein (FGSG10129), was determined by functional characterisation of deletion mutants obtained from two F. culmorum wild-type strains, FcUk99 (a highly pathogenic DON producer) and Fc233B (unable to produce toxin and with a mild pathogenic behavior). The ΔFcStuA mutants originating from both strains showed common phenotypic characters including stunted vegetative growth, loss of hydrophobicity of the mycelium, altered pigmentation, decreased activity of polygalacturonic enzymes and catalases, altered and reduced conidiation, delayed conidial germination patterns and complete loss of pathogenicity towards wheat stem base/root tissue. Glycolytic process efficiency [measured as growth on glucose as sole carbon (C) source] was strongly impaired and growth was partially restored on glutamic acid. Growth on pectin-like sources ranked in between glucose and glutamic acid with the following order (the lowest to the highest growth): beechwood xylan, sugarbeet arabinan, polygalacturonic acid, citrus pectin, apple pectin, potato azogalactan. DON production in the mutants originating from FcUK99 strain was significantly decreased (−95%) in vitro. Moreover, both sets of mutants were unable to colonise non-cereal plant tissues, i.e. apple and tomato fruits and potato tubers. No differences between mutants, ectopic and wild-type strains were observed concerning the level of resistance towards four fungicides belonging to three classes, the demethylase inhibitors epoxiconazole and tebuconzole, the succinate dehydrogenase inhibitor isopyrazam and the cytochrome bc1 inhibitor trifloxystrobin. StuA, given its multiple functions in cell regulation and pathogenicity control, is proposed as a potential target for novel disease management strategies.

Evidence for a reversible drought induced shift in the species composition of mycotoxin producing Fusarium head blight pathogens isolated from symptomatic wheat heads.

Fusarium species are fungal plant pathogens producing toxic secondary metabolites such as deoxynivalenol (DON), 15-acetyl-deoxynivalenol (15AcDON) and nivalenol (NIV). In Luxembourg, the Fusarium species composition isolated from symptomatic winter wheat heads was dominated by Fusarium graminearum sensu stricto strains (genetic 15AcDON chemotype) between the years 2009 and 2012, except for 2011, when Fusarium culmorum strains (genetic NIV chemotype) dominated the pathogen complex. Previous reports indicated that F. graminearum sensu stricto (genetic 15AcDON chemotype) was also most frequently isolated from randomly sampled winter wheat kernels including symptomatic as well as asymptomatic kernels in 2007 and 2008. The annual precipitation (average of 10 weather stations scattered across the country) decreased continuously from 924.31mm in 2007 over 917.15mm in 2008, to 843.38mm in 2009, 736.24mm in 2010, and 575.09mm in 2011. In 2012, the annual precipitation increased again to 854.70mm. Hardly any precipitation was recorded around the time of wheat anthesis in the years 2010 and 2011, whereas precipitation levels >50mm within the week preceding anthesis plus the week post anthesis were observed in the other years. The shift to genetic NIV chemotype F. culmorum strains in 2011 was accompanied by a very minor elevation of average NIV contents (2.9ngg(-1)) in the grain. Our data suggest that high NIV levels in Luxembourgish winter wheat are at present rather unlikely, because the indigenous F. culmorum strains with the genetic NIV chemotype seem to be outcompeted under humid in vivo conditions by F. graminearum DON producing strains on the one hand and seem to be inhibited - even though to a lower extent than DON producing strains - under dry in vivo conditions on the other hand.