Abstract

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Abstract

The occurrence of plant pathogens and toxigenic fungi and subsequently mycotoxin contamination in different crops around the world have significant implications for food and feed safety, food security and international trade. The main mycotoxins detected as natural contaminants in oily seeds and cereals include aflatoxins, trichothecenes, fumonisins , ochratoxin A and zearalenone. Species of Fusarium and those within Aspergillus sections Flavi and Nigri are producers of these toxic secondary metabolites. Fusarium head blight (FHB) is a devastating disease that causes extensive yield and quality losses to wheat and other small cereal grains worldwide. Different strategies including crop rotation, tillage practices, fungicide application and planting less susceptible cultivars are used in order to reduce the impact of mycotoxins in these cereal-based food and feed chains. The development of fungicide resistance together with the rising of public concern of risks associated with pesticides use has led to the search for environmentally friendly alternatives. Biocontrol offers an alternative approach that can be used in the framework of an integrated pest management (IPM) strategy to reduce the accumulation of mycotoxins in food and feed chains. Aspergillus section Flavi can infect peanuts and maize pre-harvest stage, especially during drought stress episodes resulting in aflatoxin contamination. Biocontrol based on competitive exclusion by using atoxigenic Aspergillus flavus strains is one of the most promising strategies for minimising aflatoxin contamination in both these commodities. Populations of native atoxigenic Aspergillus flavus strains were evaluated based on phenotypic, physiological and genetic characteristics. Selected atoxigenic strains of A. flavus with no capacity for aflatoxin or cyclopiazonic acid production were evaluated in field trials. Reductions of aflatoxin contamination were between 78 - 90% in treated plots in comparison with control plots. Two potential biocontrol agents, Bacillus velezensis RC 218 and Streptomyces albidoflavus RC 87B, have also been evaluated for reduction of Fusarium head blight severity and deoxynivalenol (DON) in bread and durum wheats. Both these strains effectively reduced FHB incidence (up to 30%), severity (up to 25%) and DON accumulation (up to 51%) in durum wheat under field conditions.

Abstract

Can we live without fungi? Microscopic entities or giants, friends or killers, degraders or producers, they all play crucial roles for life on our planet. Their presence has accompanied the history of humanity, but for a long time, due their largely hidden and unseen actions, their importance was not fully acknowledged and their phylogenetic relationships with animals and plants were erroneously described. Nowadays we are aware that fungi are powerful organisms, which can offer us new pharmaceuticals, help cleaning up waters and soils from contaminants, and provide crucial support to the green inhabitants of the planet. The aim of this presentation is to illustrate different strategies developed by fungi in order to beneficially interact with land plants. Fossil data reveal that fungi resembling modern Glomeromycotina were already associated with first land plants around 450 MYA. However, only today, thanks to the use of -omics technology, we can decipher their enigmatic genomes, reconstruct their metabolic pathways, describe their impact on plants, and identify the molecules involved in the dialogue occurring with their hosts. Thanks to this wealth of data, we can finally make hypotheses on the evolution and molecular mechanisms that make fungi so successful in time and space. Mycorrhizal fungi create networks not only with plants, but also with other soil inhabitants like animals, other fungi and bacteria. The dialogue with bacteria is particularly fascinating. Bacteria can live on the surface of mycelia and spores or, in a more intimate way, as endocellular symbionts inside fungal cells. In the past, the interactions between bacteria and fungi were mostly described as antagonistic in nature, however, most recent data describe cooperative activities between fungi and bacteria. Increasing attention is currently given to the concepts of microbiota and holobiont. In this context, on one hand mycorrhizal fungi are part of the plant microbiota, and represent a key component of the plant holobiont; on the other hand, they also possess their own microbiota. Thus, analogous to animals and plants, a mycorrhizal fungus may be seen at the center of a complex network of inter-kingdom interactions. An example of this tripartite symbiosis is Gigaspora margarita, an arbuscular mycorrhizal fungus that associates both with many plants and diverse endobacterial populations. Deciphering these multiple interactions will be a future goal, which may provide interesting insights into the capacity of mycorrhizal fungi to modulate their responses depending on the organism with whom they interact.

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Abstract

Cells evolved mechanisms not only to defend themselves from reactive oxygen species (ROS) but also to use ROS as growth and cell differentiation signaling molecules. Using Aspergillus nidulans as model system, we have shown that transcription factors (TF) SrrA, NapA and AtfA are individually required to survive oxidative stress and that they also regulate asexual and sexual development. Aspergillus nidulans transcription factor (TF) NapA is a member of AP-1 family, which includes fungal Yap1 and Pap1 TFs. Just like Yap1 and Pap1 orthologs, NapA accumulates in the nucleus in presence of H₂O₂, a behavior also observed in the presence of menadione, osmotic stress or glucose starvation. NapA is essential for H₂O₂ resistance and normal production of conidia, while, it represses sexual development and regulates cleistothecia pigmentation. By showing that ∆napA mutants are unable to grow in arabinose, fructose and ethanol, we uncovered a novel role for NapA in carbon utilization. This is consistent with a transcriptomic analysis showing that during conidial development NapA is required for the regulation of at least 214 genes, including ethanol utilization genes alcR, alcA and aldA, as well as other genes involved in carbohydrate utilization, transcriptional regulation, drug detoxification and secondary metabolism. Peroxiredoxins are enzymes belonging to a conserved family of peroxidases that have been involved in H₂O₂ sensing and Yap1 and Pap1 activation. The phenotypic characterization of ∆gpxA, ∆tpxA, and ∆tpxB single, double and triple peroxiredoxin mutants in wild type or ∆napA backgrounds shows that none of these Prxs is required for NapA function in H₂O₂ or menadione resistance. However, these Prxs participate in a minor NapA-independent H₂O₂ resistance pathway, while NapA and TpxA appear to regulate conidiation along the same route. While all these peroxiredoxins are not necessary for arabinose and fructose utilization, TpxA and TpxB are important for ethanol utilization, suggesting that the utilization of this carbon source involves a specific type of oxidative stress.

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Abstract

Fermented and cured meat products are unique and often represented as an element of culinary heritage and gastronomic identity. Together with meat enzymes and bacteria, molds are very important in the ripening of some dry fermented meat products. They contribute to the development of the typical sausage flavor, prevent lipid oxidations and counteract undesirable microorganisms. Various genera of fungi could colonize salami but Penicillium species are predominant, and above all P. nalgiovense, P. chrysogenum and a new recently described species P. salamii. On the other hand, depending on its peculiar composition, the surface could be colonized by undesirable molds, like P. nordicum an important and consistent producer of the potent nephrotoxic ochratoxin A (OTA). Addressing the safety of seasoning of meat products we developed different molecular approaches to detect the presence of P. nordicum and monitor OTA contamination risk. A sensitive and easy to use Loop-mediated isothermal amplification (LAMP) assay for P. nordicum detection on salami surface was set up targeting otapksPN gene, a key gene in the biosynthesis of OTA in P. nordicum. Positive reactions were detected directly in-tube by color transition of hydroxynaphthol blue from violet to sky blue. The assay was proved to be specific for P. nordicum and able to detect down to 100 fg of target DNA. In addition, gene expression of otapksPN gene in P. nordicum and OTA production were monitored throughout the seasoning process up to 30 days in a small-scale experiment. The expression of otapksPN gene was already detected after 4 days of seasoning and increased significantly after 7 days, reaching the maximum expression level after 10 days. Consistent with gene expression data, OTA was detected from the 4^th day and its content increased significantly from the 7^th day, reaching the maximum level after 10 days. Finally, the LAMP assay was tested to detect the persistence of P. nordicum during the seasoning process of sausages after co-inoculation of the fungus with P. nalgiovense at different contamination rates. After 14 days of seasoning, LAMP assay was able to detect the presence of P. nordicum down to 2.5% of P. nordicum contamination. The analysis of toxin content at the end of seasoning, revealed that OTA was accumulated both in mycelium and dry-cured meat when P. nordicum contamination rate ranged from 25% to 100% of inoculum, while OTA was not detected in dry-cured meat at 2.5% and 0.25%. These results evidenced that contamination of dry-cured meat products by P. nordicum could represent a serious concern for salami production and therefore molecular tools, such as LAMP and gene expression assay, should be considered for new HACCP plans in order to prevent and control OTA risk in dry-cured meat production.

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Abstract

Since the completion of the yeast genome, fungal genomics are leading eukaryotic genomics research advancing our knowledge in comparative genomics to pan-genomics and population genomics of fungi along with the rapid spread of sequencing technologies. The early studies of single fungal genomes told us in particular the gene contents, sex nature and particular protein families in association with fungal life styles. Pan-genome sequencing of fungal strains of the same or different species belonging to the same genus is facilitating the understanding of fungal speciation trajectory, convergence and divergence evolutions. The accumulated evidence of substantial genetic alternations existing between fungal strains promotes the sequencing/resequencing of dozens to hundreds of isolates collected from different environments for a single species. Population genomic/genetic studies of these isolates can well benefit the understanding of species origin, adaptation (including domestication), genetic selection, and phenotype-genotype associations. We are studying ascomycete insect pathogenic fungi. Genome sequencing of the model species of Metarhizium and Beauveria indicated that insect pathogens evolved with the expanded families of proteases and chitinases to target the protein- and chitin-rich insect cuticles. Species of insect pathogens can perform either sexual or asexual reproduction in nature that determines the genome structures. Comparative and phylogenomic analyses of Metarhizium species with different host ranges revealed that the generalist species evolved from the specialists via transitional species with intermediate host ranges and that this shift paralleled insect host speciation and evolution. We found that fungal host specialization was associated with the retention of sexuality and rapid evolution of existing protein sequences whereas generalization was associated with protein-family expansion, loss of genome-defense mechanisms, genome restructuring, horizontal gene transfer, and positive selection that was accelerated after reinforcement of reproductive isolation. Comparative genomic analysis with the plant and mammalian pathogens indicated that, unexpectedly, more common orthologous protein groups are shared between the insect and plant pathogens than between the insect and mammalian pathogens. We also found that the pathogenicity of host-adapted fungi evolved multiple times, and that both divergent and convergent evolution occurred during pathogen-host arms races. In particular, the effector-like proteins identified in plant and animal pathogens are highly linked to fungal host adaptation, suggesting the existence of similar gene-for-gene relationships in fungus-animal interactions that has not been established before. We also performed population genetics analysis of more than 200 field isolates of B. bassiana and revealed the population genetic diversity variations between seasons, frequent host jumping, and genetic recombination and exchanges between strains and populations. It is of importance to find that the industrial strain released for insect pest control could persist in the field and infect non-target hosts but could not displace the local populations. For different fungal species, more population genetics studies are required to help understand the nature and features of fungal adaptation to diverse environments including hosts.

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Abstract

There is interest in the impacts that climate change (CC) factors will have on the infection of staple food commodities by fungal diseases, pre-harvest and post-harvest. This applies to contamination of staple commodities with spoilage and mycotoxigenic moulds. These will have an impact on the food security agenda. CC is due to the interaction between three key environmental factors of elevated CO₂ (400 vs 800/1200 ppm), temperature increases (+2-4^oC) and drought stress. There is now some evidence that CC impacts on plant physiology including growth and yield of staple crops. We have been particularly interested in the impact that changes in CC scenarios may have on the growth/mycotoxin production by key spoilage fungi in staple food commodities. Thus, we have examined the effect of CC environmental factors on growth and mycotoxin production by Fusarium graminearum and F.langsethiae (type B and type A trichothecenes respectively), Aspergillus flavus (aflatoxins) and A.westerdijkiae and A.carbonarius (ochratoxin A). We have examined the impact that CC factors may have on growth as well as gene clusters involved in mycotoxin production. For example, by using RNAseq and information on aflatoxin B₁ production we have been able to examine the impact that such CC environmental actors may have on functioning of the biosynthesis of aflatoxins and other key secondary metabolites. Studies on mycotoxigenic Aspergillus species colonising coffee and pistachio nuts, suggest differential effects on mycotoxin contamination in vitro and in situ when exposed to CC conditions. In addition, acclimatisation to CC conditions needs to be considered. This could also have implications for the legislative limits for mycotoxins in certain food commodities. These results will be discussed in the context of the food security agenda and the implications that CC scenarios may have on the resilience of staple food crops.

Abstract

We are in golden age of fungal community ecology. An explosion in research has been driven by a combination of high-throughput sequence methods, expansive public databases, improved statistical tools, and raw computational power. Together these have allowed us to sample and analyze fungal communities at levels that were impossible just a few decades ago. Collectively this work has now provided the first global views of fungi in some of the most important guilds and ecosystems, and has allowed us to sample fungi in forms and habitats that were previously inaccessible. Along with this expansion in our knowledge of patterns has come an increasing integration of fungi into the broader field of community ecology. In particular the fit of observed pattern to theory has become a common theme within this body of work, and this focus has resulted in a greater understanding of the drivers and functional consequences fungal community structure. However, the greatest impact of fungal systems on community ecology may be in their use for testing, revising, and creating new theory. This potential is based in part on the fact that community ecology theory is heavily biased by plant ecology. This is an advantage because like plants, fungi are sessile, territorial, and frequently limited by rates of dispersal and establishment. These shared features mean that much current theory developed in plant ecology applies well to fungi. However fungi also differ from plants in the dominant types of competition employed and in the wealth of symbiotic interacts in which they are involved. Furthermore many fungal communities are much more amenable to manipulation and replication on rapid time scales than are plant or animal communities. This means that theory can often be tested more rapidly and under more controlled conditions than is possible with other types of organisms. This talk will illustrate these points with recent and historical fungal research within the broad field of community ecology.

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Abstract

Aspergillus flavus is a prevalent saprophytic and pythopathogenic fungus that causes loss of billions of dollars globally due to damage to and mycotoxin contamination of pre- and postharvest crops, and negative health effects in humans and domesticated animals. The A. flavus mycotoxins of greatest concern are the aflatoxins. Use of non-aflatoxigenic strains of A. flavus to compete against aflatoxin-producing strains has emerged as one of the best management practices for reducing aflatoxins contamination. We recently sequenced the genome and transcriptome of a new potential A. flavus biocontrol agent isolated from almond. This strain, WRRL 1519, does not produce aflatoxins or cyclopiazonic acid. The genome of WRRL 1519 was similar to other strains in size (38.0 Mb), GC content (47.2%) and number of putative proteins (12,121). Compared to aflatoxigenic A. flavus strains, strain WRRL 1519 had low shared identity or deletions for many genes and proteins required for aflatoxins and cyclopiazonic acid (CPA) syntheses. Over half of the aflatoxin synthesis gene cluster was missing, while the CPA gene cluster could not be identified. The new strain also appeared to maintain functional sequences of genes known to be involved in infectivity, particularly a pectinase gene that is thought to be required for aggressive growth in plant hosts. These results indicated that strain WRRL 1519 would be a good candidate for reducing aflatoxins and CPA accumulation by out-competing toxigenic strains in infected host crops, and warrants further experimental study. We additionally compared the genomic arrangements of predicted protein-coding genes of WRRL 1519 and other naturally-occurring biocontrol strains NRRL 21882 (Afla-Guard), NRRL 18543 (AF36) and NRRL 30797 (K49) to those of the aflatoxigenic strain NRRL 3357. While the aflatoxin synthesis gene clusters were disrupted by deletions and point mutations, our work revealed that chromosomal transpositions also appeared to disrupt several secondary metabolite gene clusters in strain WRRL 1519. The loss of secondary metabolites may affect growth rate, toxicity and effectiveness of biocontrol. Continued computational analyses and experimental work on the A. flavus genomes will identify defense, metabolic and infectivity genes of atoxigenic A. flavus strains that promote biocontrol-related management of toxin contamination.

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An unprecedented number of emerging fungal pathogens (EFPs) are emerging and causing disease in animals and plants, putting the resilience of wild and managed ecosystems in jeopardy. While the past decades have seen an increase in the number of EFPs, they have also seen the birth of new big-data technologies and analytical approaches to tackle these emerging pathogens. I explore the methodologies and bioinformatic toolkits that currently exist to rapidly analyse the genomes of unknown fungi, then discuss how these data can be used to address key questions that shed light on their epidemiology. I then show how new high-throughput experimental models, biochemical methods and informatics toolkits are allowing the fuller characterisation of ecological interactions that modify the outcome of EFPs as they occur, and speculate on future 'Big Biology' approaches that will transform our ability to tackle this increasingly important class of emerging pathogens.

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Abstract

Fungal spoilage of foods and beverages imposes significant annual global revenue losses. Mold spoilage can also be a food safety issue due to the production of mycotoxins by these molds. To prevent mold spoilage and mycotoxin production, several hurdles can be used: (1) reducing the water activity, (2) thermal processing, (3) addition of preservatives, (4) reduction of oxygen in the packaging using vacuum, oxygen scavengers or modified atmosphere packaging (MAP), and (5) refrigerated storage. These hurdles individually target a different group of spoilage fungi; the use of two or more hurdles will reduce the number of molds that can spoil the product. This is called the associated mycobiota that typically comprises only a few mold species. It is essential that the associated mycobiota be adequately isolated and accurately identified. While classic mycological detection methods can detect a broad range of fungi using well validated protocols, they are time consuming, require skilled personnel, and some methods have low sensitivity. Molecular methods for the detection of fungi from spoiled foods are faster than conventional methods but require good DNA isolation techniques, expensive equipment and may detect non-viable fungi that are unlikely to spoil a specific product. One of their advantages, especially in PCR-based methods, is the specific detection of small amounts of target organisms by amplifying their DNA in a considerably short time frame. Identification based on phenotypic characters can be time consuming and well-trained staff is needed. It is therefore more prone to erroneous identifications than a sequence-based identification. However, the results of a sequence-based identification heavily depend on the quality of the database. In order to prevent misidentifications, it is strongly recommended to use sequence-based techniques in conjunction with morphological techniques. Strain typing – distinguishing between different strains of the same species – is used to get insight in the genetic diversity of spoilage agents (is the contamination caused by the same strain?) or can be used to trace the source of the contamination. Although there is no complete or easy method for the detection of fungi in foods it is important to be aware of the limitations of each methodology. This