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.

Pathogens and insect herbivores drive rainforest plant diversity and composition.

Tropical forests are important reservoirs of biodiversity, but the processes that maintain this diversity remain poorly understood. The Janzen-Connell hypothesis suggests that specialized natural enemies such as insect herbivores and fungal pathogens maintain high diversity by elevating mortality when plant species occur at high density (negative density dependence; NDD). NDD has been detected widely in tropical forests, but the prediction that NDD caused by insects and pathogens has a community-wide role in maintaining tropical plant diversity remains untested. We show experimentally that changes in plant diversity and species composition are caused by fungal pathogens and insect herbivores. Effective plant species richness increased across the seed-to-seedling transition, corresponding to large changes in species composition. Treating seeds and young seedlings with fungicides significantly reduced the diversity of the seedling assemblage, consistent with the Janzen-Connell hypothesis. Although suppressing insect herbivores using insecticides did not alter species diversity, it greatly increased seedling recruitment and caused a marked shift in seedling species composition. Overall, seedling recruitment was significantly reduced at high conspecific seed densities and this NDD was greatest for the species that were most abundant as seeds. Suppressing fungi reduced the negative effects of density on recruitment, confirming that the diversity-enhancing effect of fungi is mediated by NDD. Our study provides an overall test of the Janzen-Connell hypothesis and demonstrates the crucial role that insects and pathogens have both in structuring tropical plant communities and in maintaining their remarkable diversity.

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.

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.

Chitosan Mediates Germling Adhesion in Magnaporthe oryzae and Is Required for Surface Sensing and Germling Morphogenesis.

The fungal cell wall not only plays a critical role in maintaining cellular integrity, but also forms the interface between fungi and their environment. The composition of the cell wall can therefore influence the interactions of fungi with their physical and biological environments. Chitin, one of the main polysaccharide components of the wall, can be chemically modified by deacetylation. This reaction is catalyzed by a family of enzymes known as chitin deacetylases (CDAs), and results in the formation of chitosan, a polymer of ß1,4-glucosamine. Chitosan has previously been shown to accumulate in the cell wall of infection structures in phytopathogenic fungi. Here, it has long been hypothesized to act as a 'stealth' molecule, necessary for full pathogenesis. In this study, we used the crop pathogen and model organism Magnaporthe oryzae to test this hypothesis. We first confirmed that chitosan localizes to the germ tube and appressorium, then deleted CDA genes on the basis of their elevated transcript levels during appressorium differentiation. Germlings of the deletion strains showed loss of chitin deacetylation, and were compromised in their ability to adhere and form appressoria on artificial hydrophobic surfaces. Surprisingly, the addition of exogenous chitosan fully restored germling adhesion and appressorium development. Despite the lack of appressorium development on artificial surfaces, pathogenicity was unaffected in the mutant strains. Further analyses demonstrated that cuticular waxes are sufficient to over-ride the requirement for chitosan during appressorium development on the plant surface. Thus, chitosan does not have a role as a 'stealth' molecule, but instead mediates the adhesion of germlings to surfaces, thereby allowing the perception of the physical stimuli necessary to promote appressorium development. This study thus reveals a novel role for chitosan in phytopathogenic fungi, and gives further insight into the mechanisms governing appressorium development in M.oryzae.

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.

Emerging fungal threats to animal, plant and ecosystem health.

The past two decades have seen an increasing number of virulent infectious diseases in natural populations and managed landscapes. In both animals and plants, an unprecedented number of fungal and fungal-like diseases have recently caused some of the most severe die-offs and extinctions ever witnessed in wild species, and are jeopardizing food security. Human activity is intensifying fungal disease dispersal by modifying natural environments and thus creating new opportunities for evolution. We argue that nascent fungal infections will cause increasing attrition of biodiversity, with wider implications for human and ecosystem health, unless steps are taken to tighten biosecurity worldwide.

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.

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.

Crop-destroying fungal and oomycete pathogens challenge food security.

Of the various crop pests and pathogens which blight our harvests, it is the fungi and oomycetes which are the most widely-dispersed groups and which lead the global invasion of agriculture. Here, we highlight the rapid growth in fungal and oomycete disease incidence and spread across the globe. We draw attention to the need for improved disease surveillance and for more sustainable agricultural intensification and consider the economic and humanitarian costs of fungal and oomycete diseases.

Economic and physical determinants of the global distributions of crop pests and pathogens.

Crop pests and pathogens pose a significant and growing threat to food security, but their geographical distributions are poorly understood. We present a global analysis of pest and pathogen distributions, to determine the roles of socioeconomic and biophysical factors in determining pest diversity, controlling for variation in observational capacity among countries. Known distributions of 1901 pests and pathogens were obtained from CABI. Linear models were used to partition the variation in pest species per country amongst predictors. Reported pest numbers increased with per capita gross domestic product (GDP), research expenditure and research capacity, and the influence of economics was greater in micro-organisms than in arthropods. Total crop production and crop diversity were the strongest physical predictors of pest numbers per country, but trade and tourism were insignificant once other factors were controlled. Islands reported more pests than mainland countries, but no latitudinal gradient in species richness was evident. Country wealth is likely to be a strong indicator of observational capacity, not just trade flow, as has been interpreted in invasive species studies. If every country had US levels of per capita GDP, then 205 ± 9 additional pests per country would be reported, suggesting that enhanced investment in pest observations will reveal the hidden threat of crop pests and pathogens.

Robust anti-oxidant defences in the rice blast fungus Magnaporthe oryzae confer tolerance to the host oxidative burst.

Plants respond to pathogen attack via a rapid burst of reactive oxygen species (ROS). However, ROS are also produced by fungal metabolism and are required for the development of infection structures in Magnaporthe oryzae. To obtain a better understanding of redox regulation in M. oryzae, we measured the amount and redox potential of glutathione (E(GSH)), as the major cytoplasmic anti-oxidant, the rates of ROS production, and mitochondrial activity using multi-channel four-dimensional (x,y,z,t) confocal imaging of Grx1-roGFP2 and fluorescent reporters during spore germination, appressorium formation and infection. High levels of mitochondrial activity and ROS were localized to the growing germ tube and appressorium, but E(GSH) was highly reduced and tightly regulated during development. Furthermore, germlings were extremely resistant to external H2O2 exposure ex planta. EGSH remained highly reduced during successful infection of the susceptible rice cultivar CO39. By contrast, there was a dramatic reduction in the infection of resistant (IR68) rice, but the sparse hyphae that did form also maintained a similar reduced E(GSH). We conclude that M. oryzae has a robust anti-oxidant defence system and maintains tight control of EGSH despite substantial oxidative challenge. Furthermore, the magnitude of the host oxidative burst alone does not stress the pathogen sufficiently to prevent infection in this pathosystem.

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.

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.

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.

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.