Fungal Tolerance Research Articles

Functional and characteristic analysis of an appressorium-specific promoter PMagas1 in Metarhizium acridum

Entomopathogenic fungi have been used as important biological control agents throughout the world. To improve the biocontrol efficacy of entomopathogenic fungi, many genes have been used to improve fungal virulence or tolerance to adverse conditions via modulating their expression with strong promoters. The Magas1 gene is specifically expressed during appressorium formation and contributes to the virulence in Metarhizium acridum. In this study, we analyzed the functional region of the promoter of Magas1 gene (PMagas1) in M. acridum using 5′-deletion technique with enhanced green fluoresces protein (EGFP) as a reporter. Results showed the full length of the PMagas1 was at least 897 bp. Two regions (-897 to −611 bp and −392 to −328 bp) were essential for the activity of PMagas1. An engineered M. acridum strain was constructed with PMagas1 driving the expression of a subtilisin-like proteinase gene Pr1A (PMagas1-PR1A). Bioassay showed that the virulence was significantly increased in PMagas1-PR1A strain compared to wild type strain. Pmagas1 promoter is suitable for the overexpression of some genes during the infection of entomopathogenic fungi, which avoids the waste of nutritional resources and the influence on other fungal characteristics during the saprophytic process of constitutive promoter.

The white-rot fungus, Phanerochaete chrysosporium, under combinatorial stress produces variable oil profiles following analysis of secondary metabolites.

The effects of combinatorial stress on lipid production in Phanerochaete chrysosporium remain understudied. This species of white-rot fungi was cultivated on solid-state media while under variable levels of known abiotic and biotic stressors to establish the effect upon fungal oil profiles. Environmental stressors induced upon the fungus included the following: temperature, nutrient limitation and interspecies competition to assess impact upon oil profiles. Fatty acid type and its concentration were determined using analytical methods of gas chromatography and mass spectrometry. Growth rate under stress was established using high-performance liquid chromatography with ergosterol as the biomarker. Fungi grown on solid-state agar were able to simultaneously produce short- and long-chain fatty acids which appeared to be influenced by nutritional composition as well as temperature. Addition of nitrogen supplements increased the growth rate, but lipid dynamics remained unchanged. Introducing competition-induced stress had significantly altered the production of certain fatty acids beyond that of the monoculture while under nutrient-limiting conditions. Linoleic acid concentrations, for example, increased from an average of 885ngμl-1 at monoculture towards 13820ngμl-1 at co-culture, following 7days of incubation. Interspecies competition produced the most notable impact on lipid production for solid-state media cultivated fungi while the addition of nitrogen supplementation presented growth and lipid accumulation to be uncorrelated. Combinatorial stress therefore influences the yield of overall lipid production as well as the number of intermediate fatty acids produced, deriving similar oil profiles to the composition of vegetable and fish oils. Fungal secondary metabolism remains highly sensitive following combinatorial stress. The outcome impacts the research towards optimizing fungal oil profiles for biomass and nutrition. Future investigations on fungal stress tolerance mechanisms need to address these environmental factors throughout the experimental design.

Foldscope: a simplified tool for the assessment of the soil fungal diversity and synthetic fertilizers tolerance

Foldscope: a simplified tool for the assessment of the soil fungal diversity and synthetic fertilizers tolerance

CaNRT2.1 Is Required for Nitrate but Not Nitrite Uptake in Chili Pepper Pathogen Colletotrichum acutatum

Chili peppers are an important food additive used in spicy cuisines worldwide. However, the yield and quality of chilis are threatened by anthracnose disease caused by Colletotrichum acutatum. Despite the impact of C. acutatum on chili production, the genes involved in fungal development and pathogenicity in this species have not been well characterized. In this study, through T-DNA insertional mutagenesis, we identified a mutant strain termed B7, which is defective for the growth of C. acutatum on a minimal nutrient medium. Our bioinformatics analysis revealed that a large fragment DNA (19.8 kb) is deleted from the B7 genome, thus resulting in the deletion of three genes, including CaGpiP1 encoding a glycosylphosphatidyl-inisotol (GPI)-anchored protein, CaNRT2.1 encoding a membrane-bound nitrate/nitrite transporter, and CaRQH1 encoding a RecQ helicase protein. In addition, T-DNA is inserted upstream of the CaHP1 gene encoding a hypothetical protein. Functional characterization of CaGpiP1, CaNRT2.1, and CaHP1 by targeted gene disruption and bioassays indicated that CaNRT2.1 is responsible for the growth-defective phenotype of B7. Both B7 and CaNRT2.1 mutant strains cannot utilize nitrate as nitrogen sources, thus restraining the fungal growth on a minimal nutrient medium. In addition to CaNRT2.1, our results showed that CaGpiP1 is a cell wall-associated GPI-anchored protein. However, after investigating the functions of CaGpiP1 and CaHP1 in fungal pathogenicity, growth, development and stress tolerance, we were unable to uncover the roles of these two genes in C. acutatum. Collectively, in this study, our results identify the growth-defective strain B7 via T-DNA insertion and reveal the critical role of CaNRT2.1 in nitrate transportation for the fungal growth of C. acutatum.

Determination of Fungal Tolerance Index to Heavy Metals and Heavy Metal Resistance Tests.

Fungal metallo-tolerance has been described in different species and plays an important role in bioremediation of contaminated environments. Metallo-tolerance is mainly documented by microdilution assays and agar well diffusion methods using equipment that can be expensive. The tolerance index can be calculated to determine the efficiency of a fungus to degrade and resist heavy metals. The present protocol is based on analyzing the tolerance index and minimum inhibitory concentration of the metallo-tolerance potential of culturable fungi on solid media. This can be calculated by daily measurements of colony size on agar supplemented with different concentrations of heavy metals. This method is an easy approach to determine fungal heavy metal resistance using simple laboratory equipment without spectroscopy.

Calcofluor white hypersensitive proteins contribute to stress tolerance and pathogenicity in entomopathogenic fungus, Metarhizium acridum.

Fungal cell wall integrity is vital for fungal pathogenesis and stress tolerance. Calcofluor white (CFW), a cell wall perturbing agent, inhibits fungal growth by binding chitin in the cell wall. The roles of CFW sensitive proteins remain insufficiently understood in pathogenic fungi. We investigated two calcofluor white hypersensitive proteins, MaCwh1 and MaCwh43, in the entomopathogenic fungus Metarhizium acridum. Both Green fluorescent protein (GFP)-tagged MaCwh1 and MaCwh43 localized at the endoplasmic reticulum. Our results showed that the ΔMacwh1 and ΔMacwh43 mutants were more sensitive to CFW and ultraviolet irradiation stress compared to wild-type and complement strains. ΔMacwh1 had a stronger sensitivity to these stresses than ΔMacwh43. Both ΔMacwh1 and ΔMacwh43 mutants showed smoother cell wall surface, and drastically reduced chitin and mannose glycoprotein level in the cell wall and glycerol level in conidia compared to wild type. Insect bioassay showed significantly attenuated virulence for both ΔMacwh1 and ΔMacwh43 mutants with impaired ability in penetrating the host cuticle. RNA-Seq analysis revealed that a large number of genes presumably involved in cell wall construction and modification, pathogenicity and stress response were down-regulated in both ΔMacwh1 and ΔMacwh43 mutants. These findings demonstrate that both Macwh1 and Macwh43 affect the fungal cell wall ultrastructure and contribute to the stress tolerance and pest control potential in M. acrdium. © 2020 Society of Chemical Industry.

Botrytis cinerea methyl isocitrate lyase mediates oxidative stress tolerance and programmed cell death by modulating cellular succinate levels

Fungi lack the entire animal core apoptotic machinery. Nevertheless, regulated cell death with apoptotic markers occurs in multicellular as well as in unicellular fungi and is essential for proper fungal development and stress adaptation. The discrepancy between appearance of an apoptotic-like regulated cell death (RCD) in the absence of core apoptotic machinery is further complicated by the fact that heterologous expression of animal apoptotic genes in fungi affects fungal RCD. Here we describe the role of BcMcl, a methyl isocitrate lyase from the plant pathogenic fungus Botrytis cinerea, in succinate metabolism, and the connection of succinate with stress responses and cell death. Over expression of bcmcl resulted in elevated tolerance to oxidative stress and reduced levels of RCD, which were associated with accumulation of elevated levels of succinate. Deletion of bcmcl had almost no effect on fungal development or stress sensitivity, and succinate levels were unchanged in the deletion strain. Gene expression experiments showed co-regulation of bcmcl and bcicl (isocitrate lyase); expression of the bcicl gene was enhanced in bcmcl deletion and suppressed in bcmcl over expression strains. External addition of succinate reproduced the phenotypes of the bcmcl over expression strains, including developmental defects, reduced virulence, and improved oxidative stress tolerance. Collectively, our results implicate mitochondria metabolic pathways, and in particular succinate metabolism, in regulation of fungal stress tolerance, and highlight the role of this onco-metabolite as potential mediator of fungal RCD.

Fungal Tolerance: An Alternative for the Selection of Fungi with Potential for the Biological Recovery of Precious Metals

The behavior of various filamentous fungi in the presence of metals such as Cu, Zn, Ni, Fe, Mn, and V has been widely reported. However, there is little information regarding metals such as Au, Ag and Pt that are not in the form of nanoparticles. The growth of eight filamentous fungi was evaluated at increasing doses of Au, Ag and Pt. The fungi were reactivated in Petri dishes with potato dextrose agar. Subsequently, individual mycelial disks from each strain were inoculated in PDA plates with the following doses of AuCl3, Ag2SO4 and PtCl4: 0, 50, 150 and 300 mg L−1, respectively. The plates were then incubated for 20 days—a period in which the diameter of the colony was measured every 24 h. Au showed the highest toxicity for the tested fungi. All silver doses decreased the growth of most of the fungi, while platinum did not cause any inhibitory effect on the growth of the eight tested fungi. With a simple test, it was possible to observe the effect of precious metals (PMs) on the growth of filamentous fungi and consider their possible biotechnological applications in the recovery of PMs from primary or secondary sources.

Heterotrimeric G-protein signalers and RGSs in Aspergillus fumigatus.

The heterotrimeric G-protein (G-protein) signaling pathway is one of the most important signaling pathways that transmit external signals into the inside of the cell, triggering appropriate biological responses. The external signals are sensed by various G-protein-coupled receptors (GPCRs) and transmitted into G-proteins consisting of the α, β, and γ subunits. Regulators of G-protein signaling (RGSs) are the key controllers of G-protein signaling pathways. GPCRs, G-proteins, and RGSs are the primary upstream components of the G-protein signaling pathway, and they are highly conserved in most filamentous fungi, playing diverse roles in biological processes. Recent studies characterized the G-protein signaling components in the opportunistic pathogenic fungus Aspergillus fumigatus. In this review, we have summarized the characteristics and functions of GPCRs, G-proteins, and RGSs, and their regulatory roles in governing fungal growth, asexual development, germination, stress tolerance, and virulence in A. fumigatus.

Calcineurin Regulates Conidiation, Chlamydospore Formation and Virulence in Fusarium oxysporum f. sp. lycopersici.

Fusarium wilt of tomato caused by the ascomycetous fungus Fusarium oxysporum f. sp. lycopersici (Fol) is widespread in most tomato planting areas. Calcineurin is a heterodimeric calcium/calmodulin-dependent protein phosphatase comprised of catalytic (Cna1) and regulatory (Cnb1) subunits. Calcineurin has been studied extensively in human fungal pathogens, but less is known about its roles in plant fungal pathogens. It is known that calcineurin regulates fungal calcium signaling, growth, drug tolerance, and virulence. However, the roles of calcineurin in Fol have not yet been characterized. In this study, we deleted calcineurin CNA1 and CNB1 genes to characterize their roles in conidiation, chlamydospore formation and virulence in Fol. Our results revealed that both cna1 and cnb1 mutants show defects in calcineurin phosphatase activity, vegetative growth and conidiation as compared to the wild type. Furthermore, calcineurin mutants exhibited blunted and swollen hyphae as observed by scanning electron microscopy. Interestingly, we found that Fol calcineurin is critical for chlamydospore formation, a function of calcineurin previously undocumented in the fungal kingdom. According to transcriptome analysis, the expression of 323 and 414 genes was up- and down-regulated, respectively, in both cna1 and cnb1 mutants. Based on the pathogen infection assay, tomato plants inoculated with cna1 or cnb1 mutant have a dramatic reduction in disease severity, indicating that calcineurin has a vital role in Fol virulence. In conclusion, our findings suggest that Fol calcineurin is required, at least in part, for phosphatase activity, vegetative growth, conidiation, chlamydospore formation, and virulence.

Isoflavonoid biosynthesis in cultivated and wild soybeans grown in the field under adverse climate conditions

The cultivation of soybean plants is one of the most important crop production sectors in the world. Isoflavones are an important defence against pathogens in soybeans. The aim of the present study was to analyse isoflavone biosynthesis in wild and cultivated soybeans grown in the field conditions in an unfavourable climate. We analysed by LCMS-IT-TOF the composition and content of isoflavonoids, productivity and fungal disease resistance of wild and cultivated. The Hefeng25 and Sfera varieties have the highest isoflavonoid content and fungal tolerance. We have shown a 3-fold increase of total isoflavonoids in Sfera, comparing with wild type, and 4- and 7-fold increases of total isoflavone aglycones in Hefeng25 and Sfera, respectively. Accordingly, the expression of genes encoding enzymes of the isoflavonoid biosynthetic pathway was also maximal in these cultivars. Thus, biosynthetic status is an important indicator of soybean productivity and resistance to pathogens in adverse climates.

Phenotypic and molecular insights into heat tolerance of formulated cells as active ingredients of fungal insecticides.

Formulated conidia of insect-pathogenic fungi, such as Beauveria and Metarhizium, serve as the active ingredients of fungal insecticides but are highly sensitive to persistent high temperatures (32-35°C) that can be beyond their upper thermal limits especially in tropical areas and during summer months. Fungal heat tolerance and inter- or intra-specific variability are critical factors and limitations to field applications of fungal pesticides during seasons favoring outbreaks of pest populations. The past decades have witnessed tremendous advances in improving fungal pesticides through selection of heat-tolerant strains from natural isolates, improvements and innovations in terms of solid-state fermentation technologies for the production of more heat-tolerant conidia, and the use of genetic engineering of candidate strains for enhancing heat tolerance. More recently, with the entry into a post-genomic era, a large number of signaling and effector genes have been characterized as important sustainers of heat tolerance in both Beauveria and Metarhizium, which represent the main species used as fungal pesticides worldwide. This review focuses on recent advances and provides an overview into the broad molecular basis of fungal heat tolerance and its multiple regulatory pathways. Emphases are placed on approaches for screening of heat-tolerant strains, methods for optimizing conidial quality linked to virulence and heat tolerance particularly involving cell wall architecture and optimized trehalose/mannitol contents, and how molecular determinants can be exploited for genetic improvement of heat tolerance and pest-control potential. Examples of fungal pesticides with different host spectra and their appropriateness for use in apiculture are given. KEY POINTS: • Heat tolerance is critical for field stability and efficacy of fungal insecticides. • Inter- and intra-specific variability exists in insect-pathogenic fungi. • Optimized production technology and biotechnology can improve heat tolerance. • Fungal heat tolerance is orchestrated by multiple molecular pathways.

Heterologous Expression of the AtNPR1 Gene in Olive and Its Effects on Fungal Tolerance.

The NPR1 gene encodes a key component of systemic acquired resistance (SAR) signaling mediated by salicylic acid (SA). Overexpression of NPR1 confers resistance to biotrophic and hemibiotrophic fungi in several plant species. The NPR1 gene has also been shown to be involved in the crosstalk between SAR signaling and the jasmonic acid-ethylene (JA/Et) pathway, which is involved in the response to necrotrophic fungi. The aim of this research was to generate transgenic olive plants expressing the NPR1 gene from Arabidopsis thaliana to evaluate their differential response to the hemibiotrophic fungus Verticillium dahliae and the necrotroph Rosellinia necatrix. Three transgenic lines expressing the AtNPR1 gene under the control of the constitutive promoter CaMV35S were obtained using an embryogenic line derived from a seed of cv. Picual. After maturation and germination of the transgenic somatic embryos, the plants were micropropagated and acclimated to ex vitro conditions. The level of AtNPR1 expression in the transgenic materials varied greatly among the different lines and was higher in the NPR1-780 line. The expression of AtNPR1 did not alter the growth of transgenic plants either in vitro or in the greenhouse. Different levels of transgene expression also did not affect basal endochitinase activity in the leaves, which was similar to that of control plants. Response to the hemibiotrophic pathogen V. dahliae varied with pathotype. All plants died by 50 days after inoculation with defoliating (D) pathotype V-138, but the response to non-defoliating (ND) strains differed by race: following inoculation with the V-1242 strain (ND, race 2), symptoms appeared after 44–55 days, with line NPR1-780 showing the lowest disease severity index. This line also showed good performance when inoculated with the V-1558 strain (ND, race 1), although the differences from the control were not statistically significant. In response to the necrotroph R. necatrix, all the transgenic lines showed a slight delay in disease development, with mean area under the disease progress curve (AUDPC) values 7–15% lower than that of the control.

Development of 2A-CHYSEL technology based multicistronic expression systems for imparting fungal tolerance

Multigene transfer is an approach for development of transgenic crops simultaneously expressing many genes.With an aim to impart necrotrophic fungal tolerance through transgenic approach, the present study describes thedevelopment of multigene construct bearing three antifungal genes as a single transcriptional unit capable of expressing at high levels and evading transgene silencing.

Effect of fungal infection and high temperature stress on antioxidant components in Brassica juncea recombinant inbred lines

Brassica juncea recombinant inbred lines (150) were assessed for enzymatic and non-enzymatic antioxidants associated with fungal diseases (Alternaria blight and white rust) and high temperature stress tolerance. The disease severity for Alternaria blight was scored under natural conditions whereas it was under artificial inoculation for white rust. Heat stress was evaluated by exposing seedlings at 45°C for 6h. No line was found to be completely resistant against Alternaria blight whereas 46 lines depicted 0% disease severity for white rust. For heat stress, 30 lines gave 100% survival. Our investigation suggested the protective role of phenylalanine and o-hydroxyphenols against Alternaria blight, glutathione against both fungal diseases and proline against high temperature stress. Significant negative correlation of phenylalanine, o-hydroxyphenols and glutathione was observed with fungal disease severity; however, proline was positively correlated to survival percentage. This information could be used by breeders in their breeding programmes for developing resistant/tolerant lines against Alternaria blight, white rust and high temperature stress.

Cadmium immobilization in aqueous solution by Aspergillus niger and geological fluorapatite.

This study investigated the application of fungus Aspergillus niger and geological fluorapatite (FAp) to cadmium (Cd) immobilization in aqueous solution. The initial Cd concentrations were set at 100, 50, 25, and 10mgL-1. The mineralogy of the products was investigated by using scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy(EDS), and attenuated total reflection-infrared spectroscopy (ATR-IR). In both A. niger + FAp + Cd and A. niger + Cd treatments, A. niger secreted abundant oxalic acid, then dissolved the FAp, and reacted with Cd2+ cations to produce relativelyinsoluble Cd oxalate. Meanwhile, FAp can provide P source to improve microbial growth. The fungal tolerance to Cd2+ was identified at around 100mgL-1. The final Cd concentrations of 13.7, 3.2, and 0.2mgL-1 were recorded for A. niger + FAp + Cd treatments with initial Cd concentrations of 50, 25, and 10mgL-1 respectively. Meanwhile, it was observed that the Cd concentration at 25mgL-1 stimulated higher bioactivities of A. niger, which further enhanced Cd bioremediation. The immobilization efficiency (%) of the treatments at low to medium Cd concentrations was in the order: Asp + FAp > Asp > FAp, while FAp alone was most efficient at the high Cd concentration of 100mgL-1. This research provides insights into the mechanisms of combining fungus and FAp as a composite to Cd contamination at various Cd levels.

The microbial community size, structure, and process rates along natural gradients of soil salinity

Over 840 M ha of arable land is affected by salinization, and the area is predicted to increase due to global change. Assessments of the status of the microbial community in saline soils have frequently been based on microbial biomass estimates, which might not accurately reflect microbial process rates in soil. Moreover, assessments of how the relative importance of major decomposer groups bacteria and fungi are affected by salinity remain inconclusive. In this study we set out to assess the soil microbial community size, structure and process rates along two salinity gradients. To distinguish between the direct effects of high salinity and indirect effects resulting from reduced soil organic matter (OM) concentrations in saline soils we also assessed the gradients following plant litter amendments to compensate for differences in OM content between soils and isolate the effect of salinity. The research aims were to (i) investigate the microbial biomass responses to salinity as estimated based on both PLFA concentrations and qPCR measurements and to compare these responses to those of respiration and microbial growth, (ii) compare the responses of bacteria and fungi to increased salinity and (iii) assess the responses of these microbial variables to the alleviation of OM scarcity expected in saline soils. Microbial biomass estimates generally were less negatively affected by salinity than bacterial growth and respiration, and were not correlated to rates of microbial growth or respiration rates. While bacterial growth was strongly inhibited by salinity, fungal growth was similar in soils of all salinities, indicating a higher fungal tolerance to salinity. OM additions increased process rates in saline soils and alleviated some of the negative impact of salinity on respiration and growth. In conclusion, this study demonstrates differential responses of saprotrophic fungi and bacteria to increasing salinity and that bacteria are directly impacted by soil salinity while fungi are responding to the indirect effect by salinity related to reduced plant C input.

The polyubiquitin gene MrUBI4 is required for conidiation, conidial germination, and stress tolerance in the filamentous fungus Metarhizium robertsii.

The polyubiquitin gene is a highly conserved open reading frame that encodes different numbers of tandem ubiquitin repeats from different species, which play important roles in different biological processes. Metarhizium robertsii is a fungal entomopathogen that is widely applied in the biological control of pest insects. However, it is unclear whether the polyubiquitin gene is required for fungal development, stress tolerance, and virulence in the entomopathogenic fungus. In the present study, the polyubiquitin gene (MrUBI4, MAA_02160) was functionally characterized via gene deletion in M. robertsii. Compared to the control strains, the MrUBI4 deletion mutant showed delayed conidial germination and significantly decreased conidial yields (39% of the wild-type 14 days post-incubation). Correspondingly, the transcript levels of several genes from the central regulatory pathways associated with conidiation, including brlA, abaA, and wetA, were significantly downregulated, which indicated that MrUBI4 played an important role in asexual sporulation. Deletion of MrUBI4 especially resulted in increased sensitivity to ultraviolet (UV) and heat-shock stress based on conidial germination analysis between mutant and control strains. The significant increase in sensitivity to heat-shock was accompanied with reduced transcript levels of genes related to heat-shock protein (hsp), trehalose, and mannitol accumulation (tps, tpp, nth, and mpd) in the MrUBI4 deletion mutant. Deletion of MrUBI4 has no effect on fungal virulence. Altogether, MrUBI4 is involved in the regulation of conidiation, conidial germination, UV stress, and heat-shock response in M. robertsii.

Copper potentiates azole antifungal activity in a way that does not involve complex formation.

To survive, fungal pathogens must acquire nutrient metals that are restricted by the host while also tolerating mechanisms of metal toxicity that are induced by the host. Given this dual vulnerability, we hypothesized that a pathogen's access to and control of essential yet potentially dangerous metal ions would affect fungal tolerance to antifungal drug stress. Here, we show that Candida albicans becomes sensitized to both Cu limitation and Cu elevation during exposure in liquid culture to the antifungal drug fluconazole, a widely prescribed antifungal agent. Spectroscopic data confirm that while fluconazole forms a complex with Cu(ii) in water, interactions of fluconazole with neither Cu(ii) nor Cu(i) are observed in the cell culture media used for the cellular assays. This result is further supported by growth assays in deletion strains that lack Cu import machinery. Overall, we establish that increases in Cu levels by as little as 40 nM over basal levels in the growth medium reduce tolerance of C. albicans to fluconazole in a way that does not require formation of a Cu-fluconazole complex. Rather, our data point to a more complex relationship between drug stress and Cu availability that gives rise to metal-mediated outcomes of drug treatment.

Fungi between extremotolerance and opportunistic pathogenicity on humans

Numerous agents of infections in humans and other mammals are found among fungi that are able to survive extreme environmental conditions and to quickly adapt to novel habitats. Nevertheless, the relationship between opportunistic potential and polyextremotolerance was not yet studied systematically in fungi. Here, the link between polyextremotolerance and opportunistic pathogenicity is shown in a kingdom-wide phylogenetic analysis as a statistically significant co-occurrence of extremotolerance (e.g. osmotolerance and psychrotolerance) and opportunism at the level of fungal orders. In addition to extremotolerance, fungal opportunists share another characteristic—an apparent lack of specialised virulence traits. This is illustrated by a comparative genomic analysis of 20 dothideomycetous and eurotiomycetous black fungi. While the genomes of specialised fungal plant pathogens were significantly enriched in known virulence-associated genes that encode secreted proteases, carbohydrate active enzyme families, polyketide synthases, and non-ribosomal peptide synthetases, no such signatures were observed in human opportunists. Together the presented results have several implications. If infection of human hosts is a side effect of fungal stress tolerance and adaptability, the human body is most likely neither the preferred habitat of such species, nor important for their evolutionary success. This defines opportunism as opposed to pathogenicity, where infection is advantageous for the species’ fitness. Since opportunists are generally incapable of the host-to-host transmission, any host-specific adaptations are likely to be lost with the resolution of the infection, explaining the observed lack of specialised virulence traits. In this scenario opportunistic infections should be seen as an evolutionary dead end and unlikely to lead to true pathogenicity.