A collection of Trichoderma isolates from natural environments in Sardinia reveals a complex virome that includes negative-sense fungal viruses with unprecedented genome organizations.

Trichoderma genus includes soil-inhabiting fungi that provide important ecosystem services in their interaction with plants and other fungi, as well as biocontrol of fungal plant diseases. A collection of Trichoderma isolates from Sardinia has been previously characterized, but here we selected 113 isolates, representatives of the collection, and characterized their viral components. We carried out high-throughput sequencing of ribosome-depleted total RNA following a bioinformatics pipeline that detects virus-derived RNA-directed RNA polymerases (RdRps) and other conserved viral protein sequences. This pipeline detected seventeen viral RdRps with two of them corresponding to viruses already detected in other regions of the world and the remaining fifteen representing isolates of new putative virus species. Surprisingly, eight of them are from new negative-sense RNA viruses, a first in the genus Trichoderma. Among them is a cogu-like virus, closely related to plant-infecting viruses. Regarding the positive-sense viruses, we report the presence of an 'ormycovirus' belonging to a recently characterized group of bisegmented single-stranded RNA viruses with uncertain phylogenetic assignment. Finally, for the first time, we report a bisegmented member of Mononegavirales which infects fungi. The proteins encoded by the second genomic RNA of this virus were used to re-evaluate several viruses in the Penicillimonavirus and Plasmopamonavirus genera, here shown to be bisegmented and encoding a conserved polypeptide that has structural conservation with the nucleocapsid domain of rhabdoviruses.

Cultivable mycoflora on bleached, decaying and healthy Posidonia oceanica leaves in a warm-edge Mediterranean location.

Marine fungi are widely distributed in the ocean, playing an important role in the ecosystems, but only little information is available about their occurrence and activity. Seagrass bleaching is also a neglected phenomenon that seems to be linked to warm environments, even though the causes are still to be defined. In this study, the cultivable mycoflora associated to the leaf conditions (bleached, necrotic and live) and section (from the base to the tip) in the seagrass Posidonia oceanica was investigated in a Mediterranean warm-edge location (Cyprus). A total of 17 Ascomycota species/taxon were identified and results highlighted that mycoflora composition changed significantly in relation to both the leaf condition and section. A few known pathogens of terrestrial plants were detected only on bleached leaves, but it remains unknown whether they have any direct connections with P. oceanica bleaching phenomenon.

Different diagnostic approaches for the characterization of the fungal community and Fusarium species complex composition of Italian durum wheat grain and correlation with secondary metabolite accumulation.

BACKGROUND: The evolution of the fungal communities associated with durum wheat was assessed using different diagnostic approaches. Durum wheat grain samples were collected in three different Italian cultivation macro-areas (north, center and south). Fungal isolation was realized by potato dextrose agar (PDA) and by deep-freezing blotter (DFB). Identification of Fusarium isolates obtained from PDA was achieved by partial tef1α sequencing (PDA + tef1α), while those obtained from DFB were identified from their morphological characteristics (DFB + mc). The fungal biomass of eight Fusarium species was quantified in grains by quantitative polymerase chain reaction (qPCR). Fungal secondary metabolites were analyzed in grains by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Correlations between Fusarium detection techniques (PDA + tef1α; DFB + mc and qPCR) and mycotoxins in grains were assessed. RESULTS: Alternaria and Fusarium showed the highest incidence among the fungal genera developed from grains. Within the Fusarium community, PDA + tef1α highlighted that F. avenaceum and F. graminearum were the most represented members, while, DFB + mc detected a high presence of F. proliferatum. Alternaria and Fusarium mycotoxins, principally enniatins, were particularly present in the grain harvested in central Italy. Deoxynivalenol was mainly detected in northern-central Italy. CONCLUSIONS: The adoption of the different diagnostic techniques of Fusarium detection highlighted that, for some species, qPCR was the best method of predicting their mycotoxin contamination in grains. © 2023 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Perfume Guns: Potential of Yeast Volatile Organic Compounds in the Biological Control of Mycotoxin-Producing Fungi.

Pathogenic fungi in the genera Alternaria, Aspergillus, Botrytis, Fusarium, Geotrichum, Gloeosporium, Monilinia, Mucor, Penicillium, and Rhizopus are the most common cause of pre- and postharvest diseases of fruit, vegetable, root and grain commodities. Some species are also able to produce mycotoxins, secondary metabolites having toxic effects on human and non-human animals upon ingestion of contaminated food and feed. Synthetic fungicides still represent the most common tool to control these pathogens. However, long-term application of fungicides has led to unacceptable pollution and may favour the selection of fungicide-resistant mutants. Microbial biocontrol agents may reduce the incidence of toxigenic fungi through a wide array of mechanisms, including competition for the ecological niche, antibiosis, mycoparasitism, and the induction of resistance in the host plant tissues. In recent years, the emission of volatile organic compounds (VOCs) has been proposed as a key mechanism of biocontrol. Their bioactivity and the absence of residues make the use of microbial VOCs a sustainable and effective alternative to synthetic fungicides in the management of postharvest pathogens, particularly in airtight environments. In this review, we will focus on the possibility of applying yeast VOCs in the biocontrol of mycotoxigenic fungi affecting stored food and feed.

Bioprospecting Phenols as Inhibitors of Trichothecene-Producing Fusarium: Sustainable Approaches to the Management of Wheat Pathogens.

Fusarium spp. are ubiquitous fungi able to cause Fusarium head blight and Fusarium foot and root rot on wheat. Among relevant pathogenic species, Fusarium graminearum and Fusarium culmorum cause significant yield and quality loss and result in contamination of the grain with mycotoxins, mainly type B trichothecenes, which are a major health concern for humans and animals. Phenolic compounds of natural origin are being increasingly explored as fungicides on those pathogens. This review summarizes recent research activities related to the antifungal and anti-mycotoxigenic activity of natural phenolic compounds against Fusarium, including studies into the mechanisms of action of major exogenous phenolic inhibitors, their structure-activity interaction, and the combined effect of these compounds with other natural products or with conventional fungicides in mycotoxin modulation. The role of high-throughput analysis tools to decipher key signaling molecules able to modulate the production of mycotoxins and the development of sustainable formulations enhancing potential inhibitors' efficacy are also discussed.

Molecular Docking and Comparative Inhibitory Efficacy of Naturally Occurring Compounds on Vegetative Growth and Deoxynivalenol Biosynthesis in Fusarium culmorum.

The fungal pathogen Fusarium culmorum causes Fusarium head blight in cereals, resulting in yield loss and contamination of the grain by type B trichothecene mycotoxins such as deoxynivalenol (DON), and its acetylated derivatives. Synthesis of trichothecenes is driven by a trichodiene synthase (TRI5) that converts farnesyl pyrophosphate (FPP) to trichodiene. In this work, 15 naturally occurring compounds that belong to the structural phenol and hydroxylated biphenyl classes were tested in vitro and in planta (durum wheat) to determine their inhibitory activity towards TRI5. In vitro analysis highlighted the fungicidal effect of these compounds when applied at 0.25 mM. Greenhouse assays showed a strong inhibitory activity of octyl gallate 5, honokiol 13 and the combination propyl gallate 4 + thymol 7 on trichothecene biosynthesis. Docking analyses were run on the 3D model of F. culmorum TRI5 containing the inorganic pyrophosphate (PPi) or FPP. Significant ligand affinities with TRI-PPi and TRI-FPP were observed for the same sites for almost all compounds, with 1 and 2 as privileged sites. Octyl gallate 5 and honokiol 13 interacted almost exclusively with sites 1 and 2, by concurrently activating strong H-bonds with common sets of amino acids. These results open new perspectives for the targeted search of naturally occurring compounds that may find practical application in the eco-friendly control of FHB in wheat.

Water distribution systems in Sardinian hospitals host invasive clonal lineages of the Fusarium oxysporum and Fusarium solani species complexes.

Several Fusarium species cause disease on human hosts, including commonly fatal infections in immunocompromised individuals. Recently, cases of hospitalized patients affected by fusaria were reported in the Tyrrhenian Island of Sardinia, Italy. To precisely characterize the Fusarium species and haplotypes present in hospitals of the region, a multilocus DNA sequence typing (MLST) approach was applied. Water distribution systems in four departments belonging to four Sardinian hospitals were sampled. Fusarium species and sequence types (STs) were identified using MLST based on sequences of the elongation factor 1-alpha (EF-1α) gene, the nuclear ribosomal DNA intergenic spacer region (IGS rDNA), and/or a portion of the second-largest subunit of RNA polymerase (RPB2) gene. The majority of isolates obtained from Sardinian hospitals (90.7%) were identified as representatives of the Fusarium oxysporum species complex (FOSC), followed by those of the F. solani species complex (FSSC) (8.2%), and F. dimerum (1.1% of all isolates). Ten STs were found among the FOSC and FSSC, with more than 60% of the isolates identified as either FOSC ST 33 or FSSC 1 (F. petroliphilum). More than half of the FOSC isolates obtained from the water systems in all four hospitals belonged to the worldwide distributed clonal lineage ST 33. This haplotype is the most prevalent among the FOSC in different countries, being responsible for the vast majority of cases of human fusariosis.

Prenylated Trans-Cinnamic Esters and Ethers against Clinical Fusarium spp.: Repositioning of Natural Compounds in Antimicrobial Discovery.

Onychomycosis is a common nail infection mainly caused by species belonging to the F. oxysporum, F. solani, and F. fujikuroi species complexes. The aim of this study was to evaluate the in vitro susceptibility of six representative strains of clinically relevant Fusarium spp. toward a set of natural-occurring hydroxycinnamic acids and their derivatives with the purpose to develop naturally occurring products in order to cope with emerging resistance phenomena. By introducing a prenylated chain at one of the hydroxy groups of trans-cinnamic acids 1-3, ten prenylated derivatives (coded 4-13) were preliminarily investigated in solid Fusarium minimal medium (FMM). Minimal inhibitory concentration (MIC) and lethal dose 50 (LD50) values were then determined in liquid FMM for the most active selected antifungal p-coumaric acid 3,3'-dimethyl allyl ester 13, in comparison with the conventional fungicides terbinafine (TRB) and amphotericin B (AmB), through the quantification of the fungal growth. Significant growth inhibition was observed for prenylated derivatives 4-13, evidencing ester 13 as the most active. This compound presented MIC and LD50 values (62-250 µM and 7.8-125 µM, respectively) comparable to those determined for TRB and AmB in the majority of the tested pathogenic strains. The position and size of the prenylated chain and the presence of a free phenol OH group appear crucial for the antifungal activity. This work represents the first report on the activity of prenylated cinnamic esters and ethers against clinical Fusarium spp. and opens new avenues in the development of alternative antifungal compounds based on a drug repositioning strategy.

Phylogenomic Analysis of a 55.1-kb 19-Gene Dataset Resolves a Monophyletic Fusarium that Includes the Fusarium solani Species Complex.

Scientific communication is facilitated by a data-driven, scientifically sound taxonomy that considers the end-user's needs and established successful practice. In 2013, the Fusarium community voiced near unanimous support for a concept of Fusarium that represented a clade comprising all agriculturally and clinically important Fusarium species, including the F. solani species complex (FSSC). Subsequently, this concept was challenged in 2015 by one research group who proposed dividing the genus Fusarium into seven genera, including the FSSC described as members of the genus Neocosmospora, with subsequent justification in 2018 based on claims that the 2013 concept of Fusarium is polyphyletic. Here, we test this claim and provide a phylogeny based on exonic nucleotide sequences of 19 orthologous protein-coding genes that strongly support the monophyly of Fusarium including the FSSC. We reassert the practical and scientific argument in support of a genus Fusarium that includes the FSSC and several other basal lineages, consistent with the longstanding use of this name among plant pathologists, medical mycologists, quarantine officials, regulatory agencies, students, and researchers with a stake in its taxonomy. In recognition of this monophyly, 40 species described as genus Neocosmospora were recombined in genus Fusarium, and nine others were renamed Fusarium. Here the global Fusarium community voices strong support for the inclusion of the FSSC in Fusarium, as it remains the best scientific, nomenclatural, and practical taxonomic option available.

Naturally Occurring Phenols Modulate Vegetative Growth and Deoxynivalenol Biosynthesis in Fusarium graminearum.

To assess the in vitro activity of five naturally occurring phenolic compounds (ferulic acid, apocynin, magnolol, honokiol, and thymol) on mycelial growth and type B trichothecene mycotoxin accumulation by Fusarium graminearum, three complementary approaches were adopted. First, a high-throughput photometric continuous reading array allowed a parallel quantification of F. graminearum hyphal growth and reporter TRI5 gene expression directly on solid medium. Second, RT-qPCR confirmed the regulation of TRI5 expression by the tested compounds. Third, liquid chromatography-tandem mass spectrometry analysis allowed quantification of deoxynivalenol (DON) and its acetylated forms released upon treatment with the phenolic compounds. Altogether, the results confirmed the activity of thymol and an equimolar mixture of thymol-magnolol at 0.5 mM, respectively, in inhibiting DON production without affecting vegetative growth. The medium pH buffering capacity after 72-96 h of incubation is proposed as a further element to highlight compounds displaying trichothecene inhibitory capacity with no significant fungicidal effect.

An endophyte of Macrochloa tenacissima (esparto or needle grass) from Tunisia is a novel species in the Fusarium redolens species complex.

Here, we report on the morphological, molecular, and chemical characterization of a novel Fusarium species recovered from the roots and rhizosphere of Macrochloa tenacissima (halfa, esparto, or needle grass) in central Tunisia. Formally described here as F. spartum, this species is a member of the Fusarium redolens species complex but differs from the other two species within the complex, F. redolens and F. hostae, by its endophytic association with M. tenacissima and its genealogical exclusivity based on multilocus phylogenetic analyses. To assess their sexual reproductive mode, a polymerase chain reaction (PCR) assay was designed and used to screen the three strains of F. spartum, 51 of F. redolens, and 14 of F. hostae for mating type (MAT) idiomorph. Genetic architecture of the MAT locus in the former two species suggests that if they reproduce sexually, it is via obligate outcrossing. By comparison, results of the PCR assay indicated that 13/14 of the F. hostae strains possessed MAT1-1 and MAT1-2 idiomorphs and thus might be self-fertile or homothallic. However, when the F. hostae strains were selfed, 11 failed to produce perithecia and one only produced several small abortive perithecia. Cirrhi with ascospores, however, were only produced by 8/28 and 4/84 of the variable size perithecia, respectively, of F. hostae NRRL 29888 and 29890. The potential for the three F. redolens clade species to produce mycotoxins, pigments, and phytohormones was assessed by screening whole genome sequence data and by analyzing extracts on cracked maize kernel cultures via liquid chromatography-mass spectrometry.

Cultivation Area Affects the Presence of Fungal Communities and Secondary Metabolites in Italian Durum Wheat Grains.

In this study, durum wheat kernels harvested in three climatically different Italian cultivation areas (Emilia Romagna, Umbria and Sardinia) in 2015, were analyzed with a combination of different isolation methods to determine their fungal communities, with a focus on Fusarium head blight (FHB) complex composition, and to detect fungal secondary metabolites in the grains. The genus Alternaria was the main component of durum wheat mycobiota in all investigated regions, with the Central Italian cultivation area showing the highest incidence of this fungal genus and of its secondary metabolites. Fusarium was the second most prevalent genus of the fungal community in all cultivation environments, even if regional differences in species composition were detected. In particular, Northern areas showed the highest Fusarium incidence, followed by Central and then Southern cultivation areas. Focusing on the FHB complex, a predominance of Fusariumpoae, in particular in Northern and Central cultivation areas, was found. Fusariumgraminearum, in the analyzed year, was mainly detected in Emilia Romagna. Because of the highest Fusarium incidence, durum wheat harvested in the Northern cultivation area showed the highest presence of Fusarium secondary metabolites. These results show that durum wheat cultivated in Northern Italy may be subject to a higher FHB infection risk and to Fusarium mycotoxins accumulation.

Clonality, spatial structure, and pathogenic variation in Fusarium fujikuroi from rain-fed rice in southern Laos.

Bakanae disease, caused by the fungal phytopathogen Fusarium fujikuroi, can be detected in most rice (Oryza sativa L.) growing areas worldwide. In this study, we investigated the population structure of this fungus in southern Lao PDR, a country located near the geographic origin of rice domestication. Microsatellites (SSRs) and mating type (MAT) analyses, pathogenicity and fungicide sensitivity tests were integrated in the study. The first key finding is that the population genetic structure of F. fujikuroi in Lao PDR is consistent with high clonal reproduction. Indeed, (i) "true" clones were identified; (ii) within populations, MAT types were frequently skewed from 1:1 ratio, (iii) linkage disequilibrium (among SSRs as also among SSRs and MAT) was present, and (iv) gene-flow between opposite MAT types within the same population is restricted. The presence of genetic divergence among areas and populations and the occurrence of positive spatial autocorrelation of genetic variation, indicate that migration is restricted, and that genetic drift plays an important role in the evolution of this fungus. Two main well-defined groups of isolates were detected (FST = 0.213) that display a non-random spatial distribution. They differ in the ability to induce seedlings death but not seedlings elongation (the typical Bakanae symptom) suggesting that the pathogen's ability to induce the two symptoms is under different genetic control. Finally, we compared two agroecosystems with contrasting characteristics: low-input and traditional (Lao PDR) vs high-input and modern (Italy). We found differences in the level of population structuring and of spatial autocorrelation. This suggests that the evolutionary potential of the fungus not only depends on its intrinsic characteristics, but is strongly influenced by other external factors, most likely by the dynamics of infested seed exchange. Thus, quarantine and chemical treatments are a way to reduce population connectivity and hence the evolutionary potential of this pathogen.

Honokiol, magnolol and its monoacetyl derivative show strong anti-fungal effect on Fusarium isolates of clinical relevance.

The antifungal activity of magnolol and honokiol, two naturally occurring hydroxylated biphenyls, and of their synthetic derivatives was evaluated on a collection of representative isolates of Fusarium oxysporum, F. solani and F. verticillioides of clinical and ecological concern. The tested compounds were proposed as a 'natural' alternative to conventional fungicides, even though a larger range of concentrations (5-400 µg/ml) was applied. The activity of magnolol and honokiol was compared with that of terbinafine (0.1-10 µg/ml), and fluconazole (1-50 µg/ml), two fungicides widely used in treating fungal infections on humans. Magnolol showed similar fungicidal activity compared to fluconazole, whereas honokiol was more effective in inhibiting mycelium growth compared to this fungicide on all tested clinical Fusarium spp. isolates. Compared to terbinafine, honokiol showed similar antifungal activity when tested on clinical F. solani isolates, whereas magnolol was less effective at all selected concentrations (5-400 µg/ml). The different position of the phenol-OH group, as well as its protection, explain different in vitro activities between magnolol, honokiol, and their derivatives. Furthermore, magnolol showed mycelium dry weight reduction at a concentration of 0.5 mM when tested on a set of agricultural isolates of Fusaria, leading to complete inhibition of some of them. Magnolol and honokiol are proposed as efficient and safe candidates for treating clinically relevant Fusaria.

A proteomic investigation of Aspergillus carbonarius exposed to yeast volatilome or to its major component 2-phenylethanol reveals major shifts in fungal metabolism.

The use of yeast-derived volatile organic compounds (VOCs) represents a promising strategy for the biological control of various plant pathogens, including mycotoxin-producing fungi. Previous studies demonstrated the efficacy of the low-fermenting yeast Candida intermedia isolate 253 in reducing growth, sporulation, and ochratoxin A biosynthesis by Aspergillus carbonarius MPVA566. This study aimed to investigate whether the inhibitory effect of the yeast volatilome is solely attributable to 2-phenylethanol, its major component, or if a synergistic effect of all volatilome components is required to achieve an effective control of the fungal growth and metabolism. Microbiological methods, HPLC measurements and a UPLC-MS/MS approach were used to investigate the metabolic profile of A. carbonarius MPVA566 at different growing conditions: standard incubation (control), exposed to C. intermedia 253 volatilome, and incubation in the presence of 2-phenylethanol. Both yeast volatilome and 2-phenylethanol succeeded in the macroscopic inhibition of the radial mycelial growth, along with a significant reduction of ochratoxin A production. Functional classification of the fungal proteome identified in the diverse growing conditions revealed a different impact of both yeast VOCs and 2-phenylethanol exposure on the fungal proteome. Yeast VOCs target an array of metabolic routes of fungal system biology, including a marked reduction in protein biosynthesis, proliferative activity, mitochondrial metabolism, and particularly in detoxification of toxic substances. Exposure to 2-phenylethanol only partially mimicked the metabolic effects observed by the whole yeast volatilome, with protein biosynthesis and proliferative activity being reduced when compared with the control samples, but still far from the VOCs-exposed condition. This study represents the first investigation on the effects of yeast-derived volatilome and 2-phenylethanol on the metabolism of a mycotoxigenic fungus by means of proteomics analysis. CHEMICAL COMPOUNDS STUDIED OR USED IN THIS ARTICLE: 2-Phenylethanol (PubChem CID: 6054); ochratoxin-A (PubChem CID: 442530); sodium dodecyl sulfate (PubChem CID: 3423265); dithiothreitol (PubChem CID: 446094); phenylmethylsulfonyl fluoride (PubChem CID: 4784); iodoacetamide (PubChem CID: 3727); ammonium bicarbonate (PubChem CID: 14013); acetic acid (PubChem CID: 176); and acetonitrile (PubChem CID: 6342).

Effect of yeast volatile organic compounds on ochratoxin A-producing Aspergillus carbonarius and A. ochraceus.

Many foods and beverages in temperate and tropical regions are prone to contamination by ochratoxin A (OTA), one of the most harmful mycotoxins for human and animal health. Aspergillus ochraceus and Aspergillus carbonarius are considered among the main responsible for OTA contamination. We have previously demonstrated that four low or non- fermenting yeasts are able to control the growth and sporulation of OTA-producing Aspergilli both in vitro and on detached grape berries: the biocontrol effect was partly due to the release of volatile organic compounds (VOCs). Aiming to further characterise the effect of VOCs produced by biocontrol yeast strains, we observed that, beside vegetative growth and sporulation, the volatile compounds significantly reduced the production of OTA by two A. carbonarius and A. ochraceus isolates. Exposure to yeast VOCs also affected gene expression in both species, as confirmed by downregulation of polyketide synthase, non-ribosomal peptide synthase, monooxygenase, and the regulatory genes laeA and veA. The main compound of yeast VOCs was 2-phenylethanol, as detected by Headspace-Solid Phase Microextraction-Gas Chromatography-Tandem Mass Spectrometry (HS-SPME-GC-MS) analysis. Yeast VOCs represent a promising tool for the containment of growth and development of mycotoxigenic fungi, and a valuable aid to guarantee food safety and quality.

Application of Low-Fermenting Yeast Lachancea thermotolerans for the Control of Toxigenic Fungi Aspergillus parasiticus, Penicillium verrucosum and Fusarium graminearum and Their Mycotoxins.

Mycotoxins are important contaminants of food and feed. In this study, low fermenting yeast (Lachancea thermotolerans) and its derivatives were applied against toxigenic fungi and their mycotoxins. A. parasiticus, P. verrucosum and F. graminearum and their mycotoxins were exposed to yeast volatile organic compounds (VOCs) and cells, respectively. VOCs reduced significantly the fungal growth (up to 48%) and the sporulation and mycotoxin synthesis (up to 96%). Very interestingly, it was shown that even 7 yeast colonies reduced Fusarium’s growth and the synthesis of its mycotoxin, deoxynivalenol (DON). Moreover, decreasing yeast nutrient concentrations did not affect the inhibition of fungal growth, but reduced DON synthesis. In addition, inactivated yeast cells were able to remove up to 82% of the ochratoxin A (OTA). As an application of these findings, the potentialities of the VOCs to protect tomatoes inoculated with F. oxysporum was explored and showed that while in the presence of VOCs, no growth was observed of F. oxysporum on the inoculated surface areas of tomatoes, in the absence of VOCs, F. oxysporum infection reached up to 76% of the tomatoes’ surface areas. These results demonstrate that the application of yeasts and their derivatives in the agriculture and food industry might be considered as a very promising and safe biocontrol approach for food contamination.

FcRav2, a gene with a ROGDI domain involved in Fusarium head blight and crown rot on durum wheat caused by Fusarium culmorum.

Fusarium culmorum is a soil-borne fungal pathogen which causes foot and root rot and Fusarium head blight on small-grain cereals, in particular wheat and barley. It causes significant yield and quality losses and results in the contamination of kernels with type B trichothecene mycotoxins. Our knowledge of the pathogenicity factors of this fungus is still limited. A transposon tagging approach based on the mimp1/impala double-component system has allowed us to select a mutant altered in multiple metabolic and morphological processes, trichothecene production and virulence. The flanking regions of mimp1 were used to seek homologies in the F. culmorum genome, and revealed that mimp1 had reinserted within the last exon of a gene encoding a hypothetical protein of 318 amino acids which contains a ROGDI-like leucine zipper domain, supposedly playing a protein-protein interaction or regulatory role. By functional complementation and bioinformatic analysis, we characterized the gene as the yeast Rav2 homologue, confirming the high level of divergence in multicellular fungi. Deletion of FcRav2 or its orthologous gene in F. graminearum highlighted its ability to influence a number of functions, including virulence, trichothecene type B biosynthesis, resistance to azoles and resistance to osmotic and oxidative stress. Our results indicate that the FcRav2 protein (and possibly the RAVE complex as a whole) may become a suitable target for new antifungal drug development or the plant-mediated resistance response in filamentous fungi of agricultural interest.

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.

Co-evolution in a landrace meta-population: two closely related pathogens interacting with the same host can lead to different adaptive outcomes.

We examined the local adaptation patterns in a system comprising several interconnected heterogeneous plant populations from which populations of two phylogenetically closely related pathogens were also sampled. The host is Hordeum vulgare (cultivated barley); the pathogens are Pyrenophora teres f. teres (net form) and Pyrenophora teres f. maculata (spot form), the causal agents of barley net blotch. We integrated two approaches, the comparison between the population structures of the host and the pathogens, and a cross-inoculation test. We demonstrated that two closely related pathogens with very similar niche specialisation and life-styles can give rise to different co-evolutionary outcomes on the same host. Indeed, we detected local adaptation for the net form of the pathogen but not for the spot form. We also provided evidence that an a-priori well-known resistance quantitative-trait-locus on barley chromosome 6H is involved in the co-evolutionary 'arms race' between the plant and the net-form pathogen. Moreover, data suggested latitudinal clines of host resistance and that different ecological conditions can result in differential selective pressures at different sites. Our data are of interest for on-farm conservation of plant genetic resources, as also in establishing efficient breeding programs and strategies for deployment of resistance genes of P. teres.