The future of fungi: threats and opportunities.

The fungal kingdom represents an extraordinary diversity of organisms with profound impacts across animal, plant, and ecosystem health. Fungi simultaneously support life, by forming beneficial symbioses with plants and producing life-saving medicines, and bring death, by causing devastating diseases in humans, plants, and animals. With climate change, increased antimicrobial resistance, global trade, environmental degradation, and novel viruses altering the impact of fungi on health and disease, developing new approaches is now more crucial than ever to combat the threats posed by fungi and to harness their extraordinary potential for applications in human health, food supply, and environmental remediation. To address this aim, the Canadian Institute for Advanced Research (CIFAR) and the Burroughs Wellcome Fund convened a workshop to unite leading experts on fungal biology from academia and industry to strategize innovative solutions to global challenges and fungal threats. This report provides recommendations to accelerate fungal research and highlights the major research advances and ideas discussed at the meeting pertaining to 5 major topics: (1) Connections between fungi and climate change and ways to avert climate catastrophe; (2) Fungal threats to humans and ways to mitigate them; (3) Fungal threats to agriculture and food security and approaches to ensure a robust global food supply; (4) Fungal threats to animals and approaches to avoid species collapse and extinction; and (5) Opportunities presented by the fungal kingdom, including novel medicines and enzymes.

Zymoseptoria tritici white-collar complex integrates light, temperature and plant cues to initiate dimorphism and pathogenesis.

Transitioning from spores to hyphae is pivotal to host invasion by the plant pathogenic fungus Zymoseptoria tritici. This dimorphic switch can be initiated by high temperature in vitro (~27 °C); however, such a condition may induce cellular heat stress, questioning its relevance to field infections. Here, we study the regulation of the dimorphic switch by temperature and other factors. Climate data from wheat-growing areas indicate that the pathogen sporadically experiences high temperatures such as 27 °C during summer months. However, using a fluorescent dimorphic switch reporter (FDR1) in four wild-type strains, we show that dimorphic switching already initiates at 15-18 °C, and is enhanced by wheat leaf surface compounds. Transcriptomics reveals 1261 genes that are up- or down-regulated in hyphae of all strains. These pan-strain core dimorphism genes (PCDGs) encode known effectors, dimorphism and transcription factors, and light-responsive proteins (velvet factors, opsins, putative blue light receptors). An FDR1-based genetic screen reveals a crucial role for the white-collar complex (WCC) in dimorphism and virulence, mediated by control of PCDG expression. Thus, WCC integrates light with biotic and abiotic cues to orchestrate Z. tritici infection.

Tackling the emerging threat of antifungal resistance to human health.

Invasive fungal infections pose an important threat to public health and are an under-recognized component of antimicrobial resistance, an emerging crisis worldwide. Across a period of profound global environmental change and expanding at-risk populations, human-infecting pathogenic fungi are evolving resistance to all licensed systemic antifungal drugs. In this Review, we highlight the main mechanisms of antifungal resistance and explore the similarities and differences between bacterial and fungal resistance to antimicrobial control. We discuss the research and innovation topics that are needed for risk reduction strategies aimed at minimizing the emergence of resistance in pathogenic fungi. These topics include links between the environment and One Health, surveillance, diagnostics, routes of transmission, novel therapeutics and methods to mitigate hotspots for fungal adaptation. We emphasize the global efforts required to steward our existing antifungal armamentarium, and to direct the research and development of future therapies and interventions.

Emerging health threat and cost of Fusarium mycotoxins in European wheat.

Mycotoxins harm human and livestock health, while damaging economies. Here we reveal the changing threat of Fusarium head blight (FHB) mycotoxins in European wheat, using data from the European Food Safety Agency and agribusiness (BIOMIN, World Mycotoxin Survey) for ten years (2010-2019). We show persistent, high, single- and multi-mycotoxin contamination alongside changing temporal-geographical distributions, indicative of altering FHB disease pressure and pathogen populations, highlighting the potential synergistic negative health consequences and economic cost.

Asynchronous development of Zymoseptoria tritici infection in wheat.

The fungus Zymoseptoria tritici causes Septoria tritici blotch of wheat. Pathogenicity begins with spore germination, followed by stomata invasion by hyphae, mesophyll colonization and fruiting body formation. It was previously found that entry into the plant via stomata occurs in a non-synchronized way over several days, while later developmental steps, such as early and late fruiting body formation, were reported to follow each other in time. This suggests synchronization of the pathogen population in planta prior to sporulation. Here, we image a fluorescent Z. tritici IPO323-derived strain during infection. We describe 6 morphologically distinct developmental stages, and determine their abundance in infected leaves, with time post inoculation. This demonstrates that 3-5 stages co-exist in infected tissues at any given time. Thus, later stages of pathogen development also occur asynchronously amongst the population of infecting cells. This merits consideration when interpreting transcriptomics or proteomics data gathered from infected plants.

Conditional promoters to investigate gene function during wheat infection by Zymoseptoria tritici.

The fungus Zymoseptoria tritici causes Septoria tritici leaf blotch, which poses a serious threat to temperate-grown wheat. Recently, we described a raft of molecular tools to study the biology of this fungus in vitro. Amongst these are 5 conditional promoters (Pnar1, Pex1A, Picl1, Pgal7, PlaraB), which allow controlled over-expression or repression of target genes in cells grown in liquid culture. However, their use in the host-pathogen interaction in planta was not tested. Here, we investigate the behaviour of these promoters by quantitative live cell imaging of green-fluorescent protein-expressing cells during 6 stages of the plant infection process. We show that Pnar1 and Picl1 are repressed in planta and demonstrate their suitability for studying essential gene expression and function in plant colonisation. The promoters Pgal7 and Pex1A are not fully-repressed in planta, but are induced during pycnidiation. This indicates the presence of inducing galactose or xylose and/or arabinose, released from the plant cell wall by the activity of fungal hydrolases. In contrast, the PlaraB promoter, which normally controls expression of an α-l-arabinofuranosidase B, is strongly induced inside the leaf. This suggests that the fungus is exposed to L-arabinose in the mesophyll apoplast. Taken together, this study establishes 2 repressible promoters (Pnar1 and Picl1) and three inducible promoters (Pgal7, Pex1A, PlaraB) for molecular studies in planta. Moreover, we provide circumstantial evidence for plant cell wall degradation during the biotrophic phase of Z. tritici infection.

Fungi, fungicide discovery and global food security.

Securing sufficient food for a growing world population is of paramount importance for social stability and the well-being of mankind. Recently, it has become evident that fungal pathogens pose the greatest biotic challenge to our calorie crops. Moreover, the loss of commodity crops to fungal disease destabilises the economies of developing nations, thereby increasing the dimension of the threat. Our best weapon to control these pathogens is fungicides, but increasing resistance puts us in an arms race against them. New anti-fungal compounds need to be discovered, such as mono-alky lipophilic cations (MALCs) described herein. Collaborations between academia and industry are imperative to establish new and efficient ways to develop these new fungicides and to bring them to the market-place.

Geometry and evolution of the ecological niche in plant-associated microbes.

The ecological niche can be thought of as a volume in multidimensional space, where each dimension describes an abiotic condition or biotic resource required by a species. The shape, size, and evolution of this volume strongly determine interactions among species and influence their current and potential geographical distributions, but the geometry of niches is poorly understood. Here, we analyse temperature response functions and host plant ranges for hundreds of potentially destructive plant-associated fungi and oomycetes. We demonstrate that niche specialization is uncorrelated on abiotic (i.e. temperature response) and biotic (i.e. host range) axes, that host interactions restrict fundamental niche breadth to form the realized niche, and that both abiotic and biotic niches show limited phylogenetic constraint. The ecological terms 'generalist' and 'specialist' therefore do not apply to these microbes, as specialization evolves independently on different niche axes. This adaptability makes plant pathogens a formidable threat to agriculture and forestry.

Threats Posed by the Fungal Kingdom to Humans, Wildlife, and Agriculture.

The fungal kingdom includes at least 6 million eukaryotic species and is remarkable with respect to its profound impact on global health, biodiversity, ecology, agriculture, manufacturing, and biomedical research. Approximately 625 fungal species have been reported to infect vertebrates, 200 of which can be human associated, either as commensals and members of our microbiome or as pathogens that cause infectious diseases. These organisms pose a growing threat to human health with the global increase in the incidence of invasive fungal infections, prevalence of fungal allergy, and the evolution of fungal pathogens resistant to some or all current classes of antifungals. More broadly, there has been an unprecedented and worldwide emergence of fungal pathogens affecting animal and plant biodiversity. Approximately 8,000 species of fungi and Oomycetes are associated with plant disease. Indeed, across agriculture, such fungal diseases of plants include new devastating epidemics of trees and jeopardize food security worldwide by causing epidemics in staple and commodity crops that feed billions. Further, ingestion of mycotoxins contributes to ill health and causes cancer. Coordinated international research efforts, enhanced technology translation, and greater policy outreach by scientists are needed to more fully understand the biology and drivers that underlie the emergence of fungal diseases and to mitigate against their impacts. Here, we focus on poignant examples of emerging fungal threats in each of three areas: human health, wildlife biodiversity, and food security.

A lipophilic cation protects crops against fungal pathogens by multiple modes of action.

The emerging resistance of crop pathogens to fungicides poses a challenge to food security and compels discovery of new antifungal compounds. Here, we show that mono-alkyl lipophilic cations (MALCs) inhibit oxidative phosphorylation by affecting NADH oxidation in the plant pathogens Zymoseptoria tritici, Ustilago maydis and Magnaporthe oryzae. One of these MALCs, consisting of a dimethylsulfonium moiety and a long alkyl chain (C18-SMe2+), also induces production of reactive oxygen species at the level of respiratory complex I, thus triggering fungal apoptosis. In addition, C18-SMe2+ activates innate plant defense. This multiple activity effectively protects cereals against Septoria tritici blotch and rice blast disease. C18-SMe2+ has low toxicity in Daphnia magna, and is not mutagenic or phytotoxic. Thus, MALCs hold potential as effective and non-toxic crop fungicides.

Threats to global food security from emerging fungal and oomycete crop pathogens.

Emerging fungal and oomycete pathogens infect staple calorie crops and economically important commodity crops, thereby posing a significant risk to global food security. Our current agricultural systems - with emphasis on intensive monoculture practices - and globalized markets drive the emergence and spread of new pathogens and problematic traits, such as fungicide resistance. Climate change further promotes the emergence of pathogens on new crops and in new places. Here we review the factors affecting the introduction and spread of pathogens and current disease control strategies, illustrating these with the historic example of the Irish potato famine and contemporary examples of soybean rust, wheat blast and blotch, banana wilt and cassava root rot. Our Review looks to the future, summarizing what we see as the main challenges and knowledge gaps, and highlighting the direction that research must take to face the challenge of emerging crop pathogens.

Many unreported crop pests and pathogens are probably already present.

Invasive species threaten global biodiversity, food security and ecosystem function. Such incursions present challenges to agriculture where invasive species cause significant crop damage and require major economic investment to control production losses. Pest risk analysis (PRA) is key to prioritize agricultural biosecurity efforts, but is hampered by incomplete knowledge of current crop pest and pathogen distributions. Here, we develop predictive models of current pest distributions and test these models using new observations at subnational resolution. We apply generalized linear models (GLM) to estimate presence probabilities for 1,739 crop pests in the CABI pest distribution database. We test model predictions for 100 unobserved pest occurrences in the People's Republic of China (PRC), against observations of these pests abstracted from the Chinese literature. This resource has hitherto been omitted from databases on global pest distributions. Finally, we predict occurrences of all unobserved pests globally. Presence probability increases with host presence, presence in neighbouring regions, per capita GDP and global prevalence. Presence probability decreases with mean distance from coast and known host number per pest. The models are good predictors of pest presence in provinces of the PRC, with area under the ROC curve (AUC) values of 0.75-0.76. Large numbers of currently unobserved, but probably present pests (defined here as unreported pests with a predicted presence probability >0.75), are predicted in China, India, southern Brazil and some countries of the former USSR. We show that GLMs can predict presences of pseudoabsent pests at subnational resolution. The Chinese literature has been largely inaccessible to Western academia but contains important information that can support PRA. Prior studies have often assumed that unreported pests in a global distribution database represent a true absence. Our analysis provides a method for quantifying pseudoabsences to enable improved PRA and species distribution modelling.

A new mechanistic model of weather-dependent Septoria tritici blotch disease risk.

We present a new mechanistic model for predicting Septoria tritici blotch (STB) disease, parameterized with experimentally derived data for temperature- and wetness-dependent germination, growth and death of the causal agent, Zymoseptoria tritici. The output of this model (A) was compared with observed disease data for UK wheat over the period 2002-2016. In addition, we compared the output of a second model (B), in which experimentally derived parameters were replaced by a modified version of a published Z. tritici thermal performance equation, with the same observed disease data. Neither model predicted observed annual disease, but model A was able to differentiate UK regions with differing average disease risks over the entire period. The greatest limitations of both models are: broad spatial resolution of the climate data, and lack of host parameters. Model B is further limited by its lack of explicitly defined pathogen death, leading to a cumulative overestimation of disease over the course of the growing season. Comparison of models A and B demonstrates the importance of accounting for the temperature-dependency of pathogen processes important in the initiation and progression of disease. However, effective modelling of STB will probably require similar experimentally derived parameters for host and environmental factors, completing the disease triangle. This article is part of the theme issue 'Modelling infectious disease outbreaks in humans, animals and plants: approaches and important themes'. This issue is linked with the subsequent theme issue 'Modelling infectious disease outbreaks in humans, animals and plants: epidemic forecasting and control'.

Rapid loss of virulence during submergence of Z. tritici asexual spores.

Zymoseptoria tritici, the causal agent of Septoria tritici blotch, is a notable pathogen of temperate-grown wheat. To better understand the mechanisms underpinning pathogenicity, leaf infection assays are commonly used to compare either the virulence of Z. tritici wildtype or mutant strains, or the susceptibility of wheat cultivars. These assays, which control for many biotic, abiotic and experimental variables, involve the application of known spore numbers to leaves. To achieve this, spore numbers are quantified during a period of aqueous suspension. Published methods rarely state the period in which spores are held in suspension, suggesting that this variable may be uncontrolled. Using simple, agar-based plating experiments, this work firstly demonstrates that blastospore culturability (the ability to form a colony when plated on appropriate agar) decreases rapidly over time during maintenance in aqueous suspension. It is subsequently shown that this reduction in culturability correlates to a reduction in the virulence of the blastospore population. This is shown in three wild type Z. tritici strains. From this, it is concluded that suspension time is a variable of major importance in experimental design and one which, if not controlled, may lead to erroneous conclusions from inter-strain comparisons. The conidia of the unrelated fungus Magnaporthe oryzae also rapidly lose culturability when stored in aqueous suspension, whereas the microspores of Fusarium oxysporum f. sp. cubense do not, suggesting that this phenomenon occurs in some but not all other fungi. Finally, a droplet method of inoculations is proposed to decrease the variability in the numbers of spores applied, within and between experiments.

Worldwide emergence of resistance to antifungal drugs challenges human health and food security.

The recent rate of emergence of pathogenic fungi that are resistant to the limited number of commonly used antifungal agents is unprecedented. The azoles, for example, are used not only for human and animal health care and crop protection but also in antifouling coatings and timber preservation. The ubiquity and multiple uses of azoles have hastened the independent evolution of resistance in many environments. One consequence is an increasing risk in human health care from naturally occurring opportunistic fungal pathogens that have acquired resistance to this broad class of chemicals. To avoid a global collapse in our ability to control fungal infections and to avoid critical failures in medicine and food security, we must improve our stewardship of extant chemicals, promote new antifungal discovery, and leverage emerging technologies for alternative solutions.

A role for random, humidity-dependent epiphytic growth prior to invasion of wheat by Zymoseptoria tritici.

Zymoseptoria tritici causes Septoria leaf blotch of wheat. The prevailing paradigm of the Z. tritici-wheat interaction assumes fungal ingress through stomata within 24-48h, followed by days of symptomless infection. This is extrapolated from studies testing the mode of fungal ingress under optimal infection conditions. Here, we explicitly assess the timing of entry, using GFP-tagged Z. tritici. We show that early entry is comparatively rare, and extended epiphytic growth possible. We test the hypotheses that our data diverge from earlier studies due to: i. random ingress of Z. tritici into the leaf, with some early entry events; ii. previous reliance upon fungal stains, combined with poor attachment of Z. tritici to the leaf, leading to increased likelihood of observing internal versus external growth, compared to using GFP; iii. use of exceptionally high humidity to promote entry in previous studies. We combine computer simulation of leaf-surface growth with thousands of in planta observations to demonstrate that while spores germinate rapidly on the leaf, over 95% of fungi remain epiphytic, growing randomly over the leaf for ten days or more. We show that epiphytic fungi are easily detached from leaves by rinsing and that humidity promotes epiphytic growth, increasing infection rates. Together, these results explain why epiphytic growth has been dismissed and early ingress assumed. The prolonged epiphytic phase should inform studies of pathogenicity and virulence mutants, disease control strategies, and interpretation of the observed low in planta growth, metabolic quiescence and evasion of plant defences by Zymoseptoria during symptomless infection.

NOXious gases and the unpredictability of emerging plant pathogens under climate change.

Emerging pathogens of crops threaten food security and are increasingly problematic due to intensive agriculture and high volumes of trade and transport in plants and plant products. The ability to predict pathogen risk to agricultural regions would therefore be valuable. However, predictions are complicated by multi-faceted relationships between crops, their pathogens, and climate change. Climate change is related to industrialization, which has brought not only a rise in greenhouse gas emissions but also an increase in other atmospheric pollutants. Here, we consider the implications of rising levels of reactive nitrogen gases and their manifold interactions with crops and crop diseases.

Emerging Fungal Threats to Plants and Animals Challenge Agriculture and Ecosystem Resilience.

While fungi can make positive contributions to ecosystems and agro-ecosystems, for example, in mycorrhizal associations, they can also have devastating impacts as pathogens of plants and animals. In undisturbed ecosystems, most such negative interactions will be limited through the coevolution of fungi with their hosts. In this article, we explore what happens when pathogenic fungi spread beyond their natural ecological range and become invasive on naïve hosts in new ecosystems. We will see that such invasive pathogens have been problematic to humans and their domesticated plant and animal species throughout history, and we will discuss some of the most pressing fungal threats of today.

Investigating chitin deacetylation and chitosan hydrolysis during vegetative growth in Magnaporthe oryzae.

Chitin deacetylation results in the formation of chitosan, a polymer of ß1,4-linked glucosamine. Chitosan is known to have important functions in the cell walls of a number of fungal species, but its role during hyphal growth has not yet been investigated. In this study, we have characterized the role of chitin deacetylation during vegetative hyphal growth in the filamentous phytopathogen Magnaporthe oryzae. We found that chitosan localizes to the septa and lateral cell walls of vegetative hyphae and identified 2 chitin deacetylases expressed during vegetative growth-CDA1 and CDA4. Deletion strains and fluorescent protein fusions demonstrated that CDA1 is necessary for chitin deacetylation in the septa and lateral cell walls of mature hyphae in colony interiors, whereas CDA4 deacetylates chitin in the hyphae at colony margins. However, although the Δcda1 strain was more resistant to cell wall hydrolysis, growth and pathogenic development were otherwise unaffected in the deletion strains. The role of chitosan hydrolysis was also investigated. A single gene encoding a putative chitosanase (CSN) was discovered in M. oryzae and found to be expressed during vegetative growth. However, chitosan localization, vegetative growth, and pathogenic development were unaffected in a CSN deletion strain, rendering the role of this enzyme unclear.

The ß-1,3-glucanosyltransferases (Gels) affect the structure of the rice blast fungal cell wall during appressorium-mediated plant infection.

The fungal wall is pivotal for cell shape and function, and in interfacial protection during host infection and environmental challenge. Here, we provide the first description of the carbohydrate composition and structure of the cell wall of the rice blast fungus Magnaporthe oryzae. We focus on the family of glucan elongation proteins (Gels) and characterize five putative ß-1,3-glucan glucanosyltransferases that each carry the Glycoside Hydrolase 72 signature. We generated targeted deletion mutants of all Gel isoforms, that is, the GH72+ , which carry a putative carbohydrate-binding module, and the GH72- Gels, without this motif. We reveal that M. oryzae GH72+ GELs are expressed in spores and during both infective and vegetative growth, but each individual Gel enzymes are dispensable for pathogenicity. Further, we demonstrated that a Δgel1Δgel3Δgel4 null mutant has a modified cell wall in which 1,3-glucans have a higher degree of polymerization and are less branched than the wild-type strain. The mutant showed significant differences in global patterns of gene expression, a hyper-branching phenotype and no sporulation, and thus was unable to cause rice blast lesions (except via wounded tissues). We conclude that Gel proteins play significant roles in structural modification of the fungal cell wall during appressorium-mediated plant infection.