Fungal Genetics and Biology

European ash dieback caused by the invasive ascomycete species Hymenoscyphus fraxineus poses the most prominent danger to common ash trees (Fraxinus excelsior) in Europe. The disease is widely distributed in Europe and currently no efficient management strategy is available. Host-induced gene silencing and exogenous dsRNA applications have shown great potential for controlling fungal diseases in crop plants. In this study, we reported in silico evidence for the presence of a functional RNA interference pathway in Hymenoscyphus fraxineus. Moreover, we showed that the transgenic expression of a double stranded RNA (dsRNA) leads to inhibition of translation of its target polyketide synthase-like gene, a fungal endogene. We explored whether the dsRNA could be introduced exogenously and demonstrated that H. fraxineus can take up externally applied dsRNA molecules. This study highlights the RNA interference mechanism in H. fraxineus and suggests exoRNA applications as a promising approach to control European ash dieback.

Fungal phytopathogens secrete extracellular vesicles (EVs) associated with enzymes and phytotoxic metabolites. While these vesicles are thought to promote infection, defining the true contents and functions of fungal EVs, as well as suitable protein markers, is an ongoing process. To expand our understanding of fungal EVs and their possible roles during infection, we purified EVs from the hemibiotrophic phytopathogen Colletotrichum higginsianum, the causative agent of anthracnose disease in multiple plant species, including Arabidopsis thaliana. EVs were purified in large numbers from the supernatant of protoplasts but not the supernatant of intact mycelial cultures. We purified two separate populations of EVs, each associated with over 700 detected proteins, including proteins involved in vesicle transport, cell wall biogenesis and the synthesis of secondary metabolites. We selected two SNARE proteins (Snc1 and Sso2) and one 14-3-3 protein (Bmh1) as potential EV markers and generated transgenic lines expressing fluorescent fusions. Each marker was confirmed to be protected inside EVs. Fluorescence microscopy was used to examine the localization of each marker during infection on Arabidopsis leaves. These findings further our understanding of EVs in fungal phytopathogens and will help build an experimental system to study EV inter-kingdom communication between plants and fungi.

BackgroundMultidrug resistance has been identified in the fungal pathogen responsible for Septoria leaf blotch, Zymoseptoria tritici, since 2011. It has been linked to the overexpression of the gene encoding the MFS1 transporter due to inserts in the promoter region of MFS1 (PMFS1), namely types I-III. Recently, two new inserts were discovered in PMFS1 that were not linked to MDR, interrogating about whether PMFS1 inserts are the only drivers of MDR in Z. tritici. The goal of our study was to gain a more complete view of MDR in Z. tritici by examining the genotypic diversity associated with the MDR phenotype in a large sample of the modern population. ResultsWe isolated 384 potential MDR strains between 2020 and 2021 in northern Europe for PMFS1 genotype and MDR assessment. We discovered six new inserts in PMFS1, bringing the total count to 13 including one insertion-deletion in the 5 UTR region. Of these, 11 display similarities with transposable elements, and 3 are not linked to MDR. Some field strains were significantly more resistant than their respective reference of the same PMFS1 genotype and some strains without insert displayed MDR phenotype. ConclusionWe described the landscape of the MDR in modern Z. tritici population and postulate that PMFS1 is a hot-spot for insertions involving transposition events. Our study shows that MDR cannot be solely explained by inserts found in PMFS1, and that additional mechanisms might be at work.

Phosphoinositides (PI) are essential components of eukaryotic membranes and function in a large number of signalling processes. While lipid second messengers are well studied in mammals and yeast, their role in filamentous fungi is poorly understood. We used fluorescent PI-binding molecular probes to localise the phosphorylated phosphatidylinositol species PI[3]P, PI[3,5]P2, PI[4]P and PI[4,5]P2 in hyphae of the endophyte Epichloe festucae in axenic culture and during interaction with its grass host Lolium perenne. We also analysed the roles of the phosphatidylinositol-4-phosphate 5-kinase MssD and the predicted phosphatidylinositol-3,4,5-triphosphate 3-phosphatase TepA, a homologue of the mammalian tumour suppressor protein PTEN. Deletion of tepA in E. festucae and in the root-infecting tomato pathogen Fusarium oxysporum had no impact on growth in culture or the host interaction phenotype. However, this mutation did uncover the presence of PI[3,4,5]P3 in septa of E. festucae and showed that TepA is required for chemotropism in F. oxysporum. The identification of PI[3,4,5]P3 in septa of{Delta} tepA strains suggests that filamentous fungi are able to generate PI[3,4,5]P3 using an alternative biosynthetic pathway and that fungal PTEN homologues are functional lipid phosphatases. The F. oxysporum chemotropism defect demonstrates a conserved role of PTEN homologues in chemotaxis across protists, fungi and mammals.

Sporothrix brasiliensis is an emerging fungal pathogen frequently associated with zoonotic transmission of sporotrichosis. Although certain virulence factors have been proposed as potential sporotrichosis determinants, the scarcity of molecular tools for reverse genetics studies on Sporothrix has significantly impeded the dissection of mechanisms underlying the disease. Here, we demonstrate that PEG-mediated protoplast transformation is a powerful method for heterologous expression in S. brasiliensis, S. schenckii and S. chilensis. Combined with CRISPR/Cas9 gene editing, this transformation protocol allowed the deletion of the putative DHN-melanin synthase gene pks1, which is a proposed virulence factor of Sporothrix species. To improve in locus integration of deletion constructs, we deleted the KU80 homologue that is critical for non-homologous end-joining DNA repair. The use of S. brasiliensis {Delta}ku80 strains enhanced homologous-directed repair during transformation resulting in increased targeted gene deletion. In conclusion, our CRISPR/Cas9-based transformation protocol provides an efficient tool for targeted gene manipulation in Sporothrix species.

Most Eukaryotic organisms produce nitric oxide (NO); however, the mechanisms underpinning NOs biosynthesis are only known in animals. In animals, there seems to be a non-described additional system for producing NO in many cell types, including blood vessels where NO is essential for blood pressure control. NO is known to be a signalling molecule of the innate immunity system in plants and fungi although no NO generation has yet been described. In the plant pathogenic fungus Fusarium graminearum, we demonstrate an extra NO-producing system in fungi that seems also present in mammals and plants and, thus, likely the evolutionary original. The discovered NO-producing enzymes are already well-known sterol-producing enzymes with more than one function. Both these enzymes are targets for statins and the major fungicides; thus, the NO production of the new system has consequences for agriculture (pathogen resistance and control) and medicine (blood pressure control, immunity and sepsis).

Secondary infections caused by the pulmonary fungal pathogen Aspergillus fumigatus are a significant cause of mortality in patients with severe Coronavirus Disease 19 (COVID-19). Even though epithelial cell damage and aberrant cytokine responses have been linked with susceptibility to COVID-19 associated pulmonary aspergillosis (CAPA), little is known about the mechanisms underpinning co-pathogenicity. Here, we analysed the genomes of 11 A. fumigatus isolates from patients with CAPA in three centres from different European countries. CAPA isolates did not cluster based on geographic origin in a genome-scale phylogeny of representative A. fumigatus isolates. Phenotypically, CAPA isolates were more similar to the A. fumigatus A1160 reference strain than to the Af293 strain when grown in infection-relevant stresses; except for interactions with human immune cells wherein macrophage responses were similar to those induced by the Af293 reference strain. Collectively, our data indicates that CAPA isolates are genomically diverse but are more similar to each other in their responses to infection-relevant stresses. A larger number of isolates from CAPA patients should be studied to identify genetic drivers of co-pathogenicity in patients with COVID-19. ImportanceCoronavirus disease 2019 (COVID-19)-associated pulmonary aspergillosis (CAPA) has been globally reported as a life-threatening complication in some patients with severe COVID-19. Most of these infections are caused by the environmental mould Aspergillus fumigatus which ranks third in the fungal pathogen priority list of the WHO. However, little is known about the molecular epidemiology of Aspergillus fumigatus CAPA strains. Here, we analysed the genomes of 11 A. fumigatus isolates from patients with CAPA in three centres from different European countries and, carried out phenotypic analyses with a view to understand the pathophysiology of the disease. Our data indicates that A. fumigatus CAPA isolates are genomically diverse but are more similar to each other in their responses to infection-relevant stresses.

The contribution of arbuscular mycorrhizal fungi (AM fungi) to plant iron (Fe) acquisition has been demonstrated in several studies. Recently, it has been shown that AM fungi use a high-affinity reductive pathway for Fe uptake. In the AM fungus Rhizophagus irregularis the ferric reductase RiFRE1 and the Fe permeases RiFTR1 and RiFTR2 have already been characterized. In an attempt to identify the third component of the reductive iron uptake pathway, a genome-wide approach has been used in R. irregularis to find genes encoding ferroxidases of the multicopper oxidase (MCO) gene family. Nine genes putatively encoding MCOs (RiMCO1-9) were identified. A phylogenetic analysis of MCO sequences of fungi from different taxonomic groups revealed that all RiMCOs clustered together in the ferroxidase/laccase group, and none with the Fet3-type ferroxidases. RiMCO1 and RiMCO3 were the only MCO genes displaying a detectable gene expression pattern typical of a high-affinity Fe transport system, indicating that RiMCO1 and RiMCO3 might have a role in the reductive high-affinity Fe uptake system. Moreover, yeast mutant complementation assays showed that the iron permease RiFTR1 can operate without the presence of a ferroxidase, indicating that it is able to transport also ferrous (II) iron.

Under certain conditions, fungi can rapidly change the size of their cells. For example, it is known that the cells of many yeast species under hyperosmosis instantly and reversibly shrink entirely, without plasmolysis, with a decrease in volume of up to 70%. There is evidence that filamentous fungi can also instantly change the diameter of their unspecialized hyphae. This property is fundamental but requires detailed study. In this large-scale study (involving more than 50,000 cells measured) using light microscopy, the ability of three unrelated basidiomycete species to rapidly change the diameter of their hyphae under various factors was analyzed. It was found that all three fungi respond similarly to moderate hyperosmotic shock or detergent treatment, shrinking by an average of 12-14% in diameter. However, inhibitors of actin assembly can cause either expansion or shrinkage of hyphae or have no effect on a fungus. These results, along with previously established features of the macroinvagination systems of the plasma membrane in basidiomycetes, are important for understanding the complex structural-protective physiological mechanisms responsible for the survival and continuous functioning of fungal cells in unstable environmental conditions.

Apple production in Sweden and elsewhere is being threaten by the fungus, Neonectria ditissima, which causes a disease known as Fruit Tree Canker. The disease can cause extensive damages and the removal of diseased-wood and heavily infected trees can be laborious and expensive. Currently, there is no way to eradicate the fungus from infected trees and our knowledge of the infection process is limited. Thus, in order to target and modify genes efficiently, the genetic transformation technique developed for N. ditissima back in 2003 was modified. We report on the upgraded protocol and show that protoplasts were viable, able to uptake foreign DNA, and able to regenerate back into a mycelial colony, either as targeted gene-disruption mutants or as ectopic mutants expressing GFP.

Botrytis cinerea is a major pathogen of more than 1400 plant species. During infection, the kills host cells during infection and spreads through necrotic tissue, which is believed to be supported by induction of programmed plant cell death. To comprehensively evaluate the contributions of most of the currently known plant cell death inducing proteins (CDIPs) and metabolites for necrotrophic infection, an optimized CRISPR/Cas protocol was established which allowed serial marker-free mutagenesis to generate Botrytis mutants lacking up to 12 different CDIPs. Infection analysis revealed a decrease in virulence with increasing numbers of knockouts, and differences in the effects of knockouts on different host plants. The on planta secretomes obtained from these mutants revealed substantial remaining necrotic activity after infiltration into leaves. Our study has addressed for the first time the functional redundancy of virulence factors of a fungal pathogen, and demonstrates that B. cinerea releases a highly redundant cocktail of proteins and metabolites to achieve necrotrophic infection of a wide variety of host plants.

Hyphal fusion and sexual development in filamentous fungi rely on coordinated signaling of numerous conserved nodes such as the striatin interacting phosphatase and kinase (STRIPAK) complex or the pheromone response (PR) MAP kinase cascade (MIK2, MEK2, MAK2, HAM5). Here we used the homothallic ascomycete Sordaria macrospora (Sm) to screen for putative protein interactors of the SmSTRIPAK complex. Using the STRIPAK complex interactor 1 (SCI1) subunit of the complex as bait, we enriched and identified canonical SmSTRIPAK components and a determinant of communication (DOC) protein. The DOC proteins were previously described in the closely related and heterothallic species Neurospora crassa, functioning in allorecognition of germlings and hyphal fusions. We generated {Delta}Smdoc1, {Delta}Smdoc2 single deletion strains and the double deletion mutant {Delta}Smdoc1{Delta}Smdoc2 in S. macrospora. Deletion phenotypes were paradoxical: single knockouts ({Delta}Smdoc1 or {Delta}Smdoc2) were nearly sterile and sexual development was impaired, yet the double mutant ({Delta}Smdoc1{Delta}Smdoc2) exhibited wild-type fertility and development, demonstrating non-redundant and mutually antagonistic roles. Similarly, we demonstrated an impairment of the N. crassa {Delta}doc-2 mutant in sexual development. Using gene tagging at the native locus, we performed TurboID-based proximity mapping with SmDOC1 and SmDOC2 as bait proteins. This proximity mapping demonstrated close ties of SmDOC1/2 to components of the PR MAP kinase pathway and revealed mutual SmDOC1 - SmDOC2 proximity. Yeast Two-Hybrid experiments with SmDOC1 confirmed the direct interaction with the MAP kinases MEK2 and MAK2. Fluorescence microscopy revealed that SmDOC1-TagRFP-T localized to ring-like structures around septal pores. Our results demonstrate that the DOC system is not restricted to heterothallic N. crassa, but also plays an essential role in the development of fruiting bodies in the homothallic fungus S. macrospora. These findings suggest the DOC1/2 proteins as a novel system that integrates STRIPAK and PR pathways, providing a possible mechanistic explanation for their non-additive deletion strain phenotypes.

It has been hypothesized that AT-biased genotypes of Ophiocordyceps sinensis are generated through repeat-induced point mutation (RIP) and coexist as permanently nonfunctional internal transcribed spacer (ITS) pseudogenes in the genome of Hirsutella sinensis (GC-biased Genotype #1 of O. sinensis). This study examined the H. sinensis genome, which contains multiple repetitive ITS copies (GC content: 64.7{+/-}0.33%) with multiple insertion/deletion and transversion alleles, which were not generated through RIP mutagenesis that theoretically causes cytosine-to-thymine (C-to-T) and guanine-to-adenine (G-to-A) transitions. The repetitive ITS copies in the H. sinensis genome were found to be genetically and phylogenetically distinct from the AT-biased O. sinensis genotypes (GC content: 51.1{+/-}1.69%), which possess multiple transition alleles. The sequences of Genotypes #2-17, both GC- and AT-biased, are absent from the H. sinensis genome; these genotypes belong to interindividual O. sinensis fungi and differentially occur in different compartments of natural Cordyceps sinensis, with dynamic alterations in abundance occurring in an asynchronous, disproportional manner during C. sinensis maturation. Metatranscriptomic analyses of natural C. sinensis revealed the transcriptional silencing of 5.8S genes in all C. sinensis- colonizing fungi, including H. sinensis. The transcription assay reported by Li et al. [1] provided unsound, controversial evidence indicating that the 5.8S genes of AT-biased genotypes are nonfunctional pseudogenes. In addition to the single ITS locus analysis, repetitive genomic copies were also examined at multiple loci in the H. sinensis genome, and approximately 8.2% of the authentic genes had repetitive copies, including various transitions, transversions, and insertions/deletions. The transcripts for the repetitive copies, regardless of the decreases, increases, or bidirectional changes in the AT content, were identified in the H. sinensis transcriptome. These results are inconsistent with those of RIP mutagenesis, which generates pseudogenic, nonfunctional, repetitive copies. In conclusion, AT-biased genotypes of O. sinensis might have evolved through evolutionary mechanisms from a common ancestor over the long course of evolution, in parallel with GC-biased Genotype #1 H. sinensis.

Fungal species are typically either fully self-fertile or self-sterile, but some filamentous ascomycetes can commonly transition from self-fertility to self-sterility through unidirectional mating-type switching. In these fungi, the structure of the mating-type (MAT1) locus governs sexual behaviour: MAT-2 self-fertile individuals retain both MAT1-1 and MAT1-2 genes, while MAT-1 self-sterile isolates lose MAT1-2 genes during switching. A third type of isolate morphology also occurs under laboratory conditions: these are self-sterile isolates which retain both MAT1-1 and MAT1-2, but are unable to switch mating type. These are commonly referred to as MAT-2 self-sterile isolates. Two of the mating-type (MAT) genes, one of which is deleted during switching, encode transcription factors known to regulate not only the sexual cycle but also genes unrelated to mating. To test how MAT1 structural variations affects gene expression, we studied Ceratocystis albifundus, a species that switches mating type. To minimise variability caused by intraspecific genetic differences, two self-sterile isolates (MAT-1 and MAT-2 self-steriles) were derived from the same MAT-2 self-fertile parent, making all three isolates genetically identical except at the MAT1 locus. Comparative transcriptomic analyses revealed that the MAT-2 self-fertile, MAT-1 self-sterile and MAT-2 self-sterile isolates all exhibited distinct expression patterns, including differences in MAT genes, the pheromone-receptor pathway, and other genes not directly linked to mating. The results show that MAT1 locus structure influences gene expression more broadly than those only related to the sexual cycle.

Plant pathogenic fungi have evolved different strategies to interfere with plant defense mechanisms. The well described fungal plant pathogen Fusarium graminearum is not only able to produce trichothecene toxins like deoxynivalenol, but also the plant hormone auxin. Highly elevated levels of auxin and auxin derivatives such as IAA-glucoside or IAA amino-acid conjugates were observed in wheat cultivar Apogee infected with F. graminearum. We report that F. graminearum is able to cleave tryptamine-derived hydroxycinnamic acid amides, e.g. the defense compound coumaroyl-tryptamine. In this study we investigated copper amine-oxidases, candidate genes for auxin biosynthesis converting tryptamine into the IAA precursor indole-3-acetyldehyde. After consecutive knock outs of all seven copper amine oxidases the resulting septuple knock out strain had strongly reduced ability to produce auxin. Virulence of the septuple mutant was significantly impaired while DON production in planta was comparable to the wild type. We conclude that F. graminearum, often presumed to be a simple nectrotroph, has a biotrophic phase and is able to employ plant defense compounds by converting them into defense suppressing auxin.

Amidophosphoribosyl transferase catalyzes the first step of the purine nucleotide biosynthesis by converting 5-phosphoribosyl-1-pyrophosphate into 5-phosphoribosyl-1-amine. In this study, we identified and characterized the functions of MoAde4, an ortholog of yeast Ade4 in the rice blast fungus. MoAde4 is a 537-amino acid protein containing the GATase_6 and pribosyltran domains. Quantitative real-time PCR analysis showed MoADE4 transcripts were highly expressed during conidiation, early-infection, and late-infection stages of the fungus. Disruption of MoADE4 gene resulted in {Delta}Moade4 mutant exhibiting adenine, adenosine, and hypoxanthine auxotrophy on MM. Conidia quantification assays showed {Delta}Moade4 mutant was significantly reduced in sporulation. The conidia of {Delta}Moade4 mutant could still form appressoria but mostly failed to penetrate the rice cuticle. Pathogenicity test showed {Delta}Moade4 was completely nonpathogenic on rice and barley leaves which was attributed by failure of its infectious hyphae to colonize the host cells. The {Delta}Moade4 was defective in induction of strong host immunity and had its purine transporter genes repressed during in planta infection. Addition of exogenous adenine partially rescued conidiation and pathogenicity defects of the {Delta}Moade4 mutant on the barley and rice leaves. Localization assays showed that MoAde4 is located in the cytoplasm. Taken together, our results demonstrate that purine biosynthesis orchestrated by MoAde4 is required for fungal development, conidiation, more importantly, we found it to be essential for fungal pathogenicity not because of the appressorial formation, but appressorium penetration and host colonization during the plant infection of M. oryzae. Thus this findings suggests that purine biosynthesis could act as an important target for combating recalcitrant plant fungal pathogens.

Cre1 is an important transcription factor that regulates carbon catabolite repression (CCR) and is widely conserved across fungi. This gene has been extensively studied in several Ascomycota species, whereas its role in gene expression regulation in the Basidiomycota remains poorly understood. Here, we identified and investigated the role of cre1 in Coprinopsis cinerea, a basidiomycete model mushroom that can efficiently degrade lignocellulosic plant wastes. We used a rapid and efficient gene deletion approach based on PCR-amplified split-marker DNA cassettes together with in-vitro assembled Cas9-guide RNA ribonucleoproteins (Cas9-RNPs) to generate C. cinerea cre1 gene deletion strains. Gene expression profiling of two independent C. cinerea cre1 mutants showed significant deregulation of carbohydrate metabolism, plant cell wall degrading enzymes (PCWDEs), plasma membrane transporter-related and several transcription factor encoding genes, among others. Our results support the notion that, similarly to reports in the ascomycetes, Cre1 of C. cinerea orchestrates CCR through a combined regulation of diverse genes, including PCWDEs, transcription factors that positively regulate PCWDEs and membrane transporters which could import simple sugars that can induce the expression of PWCDEs. Somewhat paradoxically, though in accordance with other Agaricomycetes, genes related to lignin degradation were mostly downregulated in cre1 mutants, indicating they fall under different regulation than other PCWDEs. The gene deletion approach and the data presented in this paper expand our knowledge of CCR in the Basidiomycota and provide functional hypotheses on genes related to plant biomass degradation. ImportanceMushroom-forming fungi include some of the most efficient degraders of lignocellulosic plant biomass. They degrade dead plant materials by a battery of lignin-, cellulose-, hemicellulose- and pectin-degrading enzymes, the encoding genes of which are under tight transcriptional control. One of the highest-level regulation of these metabolic enzymes is known as carbon catabolite repression, which is orchestrates by the transcription factor Cre1, and ensures that costly lignocellulose-degrading enzyme genes are expressed only when simple carbon sources (e.g. glucose) are not available. Here, we identified the Cre1 ortholog in a litter-decomposer Agaricomycete, Coprinopsis cinerea, knocked it out and characterized transcriptional changes in the mutants. We identified several dozen lignocellulolytic enzyme genes as well as membrane transporters and other transcription factors as putative target genes. These results extend knowledge on carbon catabolite repression to litter decomposer Basidiomycota.

The fungal pathogen Parastagonospora nodorum utilises necrotrophic effectors (NEs) to cause chlorosis and necrosis on wheat. NE expression is mediated by an assortment of transcription factors (TF), the most well-characterised of which is PnPf2. Orthologues of PnPf2 regulate virulence in phytopathogenic fungi across the Ascomycete fungal lineage, yet their protein architecture remains functionally uncharacterised. These orthologues are characterised by the archetypal N-terminal Zn2Cys6 zinc-finger DNA-binding domain (DBD), a conserved yet poorly characterised middle-homology region (MHR) and a C-terminal disordered region. We investigated the role of each of the three domains through PnPf2 truncation mutants in situ. This revealed the conserved MHR of PnPf2 is required for the development of disease symptoms on wheat, but also that the C-terminal disordered region in-part modulates NE expression. Domain-interaction analysis through yeast-2-hybrid (Y2H) screening reveals PnPf2 forms a homodimer mediated by the MHR, indicating its importance mediating protein-protein interactions. Using the MHR as a bait in library-scale protein-protein interaction Y2H assays, we identified the COP9-signalosome protein PnCsn6 as a key interaction partner. PnCsn6 is essential for disease symptoms during P. nodorum infection on wheat, including NE expression. Our study presents the first domain-level functional investigation this virulence-regulating TF orthologue, the results of which underpin the essential role of the MHR in driving protein-protein interactions and effector regulation. It also reveals an essential protein-signalling pathway with which PnPf2 directly interacts and shares regulatory control of virulence.

Accurate taxonomic classification of samples from infected host material is essential for disease diagnostics and genome analyses. Despite the importance, diagnosis of fungal pathogens causing banana leaf diseases remains challenging. Foliar diseases of bananas are mainly caused by three Pseudocercospora species, of which the most predominant causal agent is P. fijiensis. Here, we sequenced and assembled four fungal isolates obtained from necrotic banana leaves in Bohol (Philippines) and obtained a high-quality genome assembly for one of these isolates. The samples were initially identified as P. fijiensis using PCR diagnostics, however, the assembly size was consistently 30 Mb smaller than expected. Based on the ITS sequences, we identified the samples as Zasmidium syzygii (98.7% identity). The high-quality Zasmidium syzygii assembly is 42.5 Mb in size, comprising 16 contigs, of which 11 are complete. The genome contains 98.6% of the expected single-copy BUSCO genes and contains 14,789 genes and 10.3% repeats. The three short-read assemblies are less continuous but have similar genome sizes (40.4 - 42.4 Mb) and contain between 96.5% and 98.4% BUSCO genes. All four isolates have identical ITS sequences and are distinct from Zasmidium isolates that were previously sampled from banana leaves. We thus report the first continuous genome assembly of a member of the Zasmidium genus, forming an essential resource for further analysis to enhance our understanding of the diversity of pathogenic fungal isolates as well as fungal diversity.

Leaf mold caused by the ascomycete fungus Cladosporium fulvum is a devastating disease of tomato plants. The mycoparasitic fungus Hansfordia pulvinata is an effective biocontrol agent that parasitizes C. fulvum hyphae on leaves and secretes 13-deoxyphomenone, an eremophilane-type sesquiterpene, which was also identified as a sporulation-inducing factor in Aspergillus oryzae. Here, we identified deoxyphomenone biosynthesis (DPH) gene clusters conserved in both H. pulvinata and Aspergillus section Flavi including A. oryzae and A. flavus. Functional disruption of DPH1 orthologous genes encoding sesquiterpene cyclase in H. pulvinata, A. oryzae and its close relative A. flavus revealed that deoxyphomenone in H. pulvinata had exogenic antifungal activity against the host fungus C. fulvum and controlled endogenic sporulation in Aspergillus species. Deoxyphomenone also inhibited mycelial growth of C. fulvum and the non-host tomato pathogen Pseudocercospora fuligena. Complete DPH clusters, highly similar to those in H. pulvinata, were exclusive to Aspergillus section Flavi, while species in other Aspergillus sections contained fragmented DPH clusters. A comparative genomics analysis revealed that these DPH gene clusters share a common origin and are horizontally transferred across large taxonomic distances from an ancestor of Aspergillus to H. pulvinata. Our results suggest that, after horizontal transfer, H. pulvinata maintained the DPH cluster as the inhibitory effect of deoxyphomenone on spore germination and mycelial growth contributed to its mycoparasitism on the host fungus C. fulvum.