Extracellular vesicles (EVs) are tiny messengers that convey bioactive molecules from donor to recipient cells, leading to changes in their physiology and function. We investigated the role of EVs in shaping growth and the biofilm biology of the tree pathogen Fusarium circinatum and its interaction with the susceptible host, Pinus patula. Vesicles were collected from fungal planktonic and biofilm cultures and from pine seedling needles and roots. The physical properties of these vesicles were analysed using nanoparticle tracking analysis and transmission electron microscopy, which revealed a diverse range of sizes and shapes, respectively. Furthermore, uptake of vesicles by conidia was conducted. The results demonstrated that F. circinatum EVs significantly but variably affected spore viability during the early phase (2-4 h) although they enhanced fungal biofilm integrity. In contrast, P. patula EVs greatly inhibited hyphal formation and biofilm biomass, but failed to inhibit matrix production in the fungal biofilm. Our results therefore show that conidial germination is essential for late fungal development including hyphal and biofilm formation while matrix production is a counter measure against harsh environmental conditions including the effects of plant-derived EVs.

Diplodia sapinea (Dothideomycetes) is a latent fungal pathogen with a global distribution that predominantly infects Pinus species. The impact of the fungus is increasing due to climate-driven range expansion and thus wide-scale disease outbreaks are occurring. With the aim of developing high-quality genome resources, we generated chromosome-level genome assemblies for 3 D. sapinea isolates and low-coverage Illumina genome data for 6 additional isolates. By comparing these genome assemblies, we identified 14 core chromosomes and 2 accessory chromosomes (ACs) in the pathogen. These 2 ACs encode 80 and 147 proteins, respectively, while 11,374 to 11,601 genes were identified in the core chromosomes. Both ACs had lower gene density and higher proportions of transposable elements compared to the core chromosomes. Sequence analysis indicated that genes on the ACs displayed more sequence variation compared to those on the core chromosomes, suggesting they serve as evolutionary hotspots in the species. Sequence homology analyses suggested that the ACs were possibly acquired horizontally, probably from a species in the Dothideomycetes. We designed PCR-based assays to detect the presence of ACs and applied these on a set of 37 isolates from 14 countries. One of the ACs was detected in 33 isolates from 13 countries, while the other AC was absent in all isolates tested. Pathogenicity trials on Pinus patula seedlings showed no correlation between the presence of ACs and isolate aggressiveness. The high-quality genomes provided here offer important resources for future research on this globally important pathogen, including the biological roles of the ACs.

The genus Elsinoe contains many aggressive pathogens of a wide range of plants, many of which are economically important. These fungal pathogens cause serious scab diseases affecting various plant parts, impacting plant vigour, yield and market value. While studies on Elsinoe species have predominantly focused on their taxonomy, there is a conspicuous gap in knowledge of these fungi from a plant pathology perspective. In this review, we draw together and critically evaluate the existing, but rather fragmented, research on the taxonomic status, phylogenetic relationships, host range, as well as the biology and epidemiology of Elsinoe species. Our aim is primarily to augment the existing understanding of the global significance of Elsinoe species, and furthermore, to shed light on the escalating prominence of scab diseases caused by species in a fungal genus that has been known for over 100 years but remains relatively poorly understood and somewhat enigmatic.

Sexual reproduction in basidiomycete fungi is governed by MAT loci (P/R and HD), which exhibit remarkable evolutionary plasticity, characterized by expansions, rearrangements, and gene losses often associated with mating system transitions. The sister genera Cryptococcus and Kwoniella provide a powerful framework for studying MAT loci evolution owing to their diverse reproductive strategies and distinct architectures, spanning bipolar and tetrapolar systems with either linked or unlinked MAT loci. Building on recent comparative genomic analyses, we generated additional chromosome-level assemblies, uncovering distinct trajectories shaping MAT loci organization. Contrasting with the small-scale expansions and gene acquisitions observed in Kwoniella, our analyses revealed independent expansions of the P/R locus in tetrapolar Cryptococcus, possibly driven by pheromone gene duplications. Notably, these expansions coincided with a pronounced GC-content reduction best explained by reduced GC-biased gene conversion following recombination suppression, rather than relaxed codon usage selection. Diverse modes of MAT locus linkage were also identified, including three previously unrecognized transitions: one resulting in a pseudobipolar arrangement and two leading to bipolarity. All three transitions involved translocations. In the pseudobipolar configuration, the P/R and HD loci remained on the same chromosome but genetically unlinked, whereas the bipolar transitions additionally featured rearrangements that fused the two loci into a nonrecombining region. Mating assays confirmed a sexual cycle in Cryptococcus decagattii, demonstrating its ability to undergo mating and sporulation. Progeny analysis in Kwoniella mangrovensis revealed substantial ploidy variation and aneuploidy, likely stemming from haploid-diploid mating, yet evidence of recombination and loss of heterozygosity indicates that meiotic exchange occurs despite irregular chromosome segregation. Our findings underscore the importance of continued diversity sampling and provide further evidence for convergent evolution of fused MAT loci in basidiomycetes, offering new insights into the genetic and chromosomal changes driving reproductive transitions.

The chestnut blight pathogen Cryphonectria parasitica is well-known for the devastation it caused to North American forests. It is less well recognized that numerous other fungi in the Cryphonectriaceae are emerging as threats to native and planted forests in the tropics and Southern Hemisphere. Unlike C. parasitica, these fungi, such as Chrysoporthe cubensis, initially gained attention due to a canker disease in plantations of non-native Eucalyptus. More than four decades of research have revealed a wide diversity of Cryphonectriaceae species that infect many other tree genera in the Myrtales. These fungi often exist as endophytes but become problematic when trees are planted outside their native range. Growing numbers of species are also undergoing host shifts from native to susceptible trees such as Eucalyptus, posing serious risks to both natural and planted forests. These fungi provide an example of the biodiversity of tree-infecting fungi that is understudied, despite their significant potential to harm forest ecosystems.

Sexual reproduction in basidiomycete fungi is governed by MAT loci (P/R and HD), which exhibit remarkable evolutionary plasticity, characterized by expansions, rearrangements, and gene losses often associated with mating system transitions. The sister genera Cryptococcus and Kwoniella provide a powerful framework for studying MAT loci evolution owing to their diverse reproductive strategies and distinct architectures, spanning bipolar and tetrapolar systems with either linked or unlinked MAT loci. Building on recent comparative genomic analyses, we generated additional chromosome-level assemblies, uncovering distinct trajectories shaping MAT loci organization. Contrasting with the small-scale expansions and gene acquisitions observed in Kwoniella, our analyses revealed independent expansions of the P/R locus in tetrapolar Cryptococcus, possibly driven by pheromone gene duplications. Notably, these expansions coincided with a pronounced GC-content reduction best explained by reduced GC-biased gene conversion following recombination suppression, rather than relaxed codon usage selection. Diverse modes of MAT locus linkage were also identified, including three previously unrecognized transitions: one resulting in a pseudobipolar arrangement and two leading to bipolarity. All three transitions involved translocations. In the pseudobipolar configuration, the P/R and HD loci remained on the same chromosome but genetically unlinked, whereas the bipolar transitions additionally featured rearrangements that fused the two loci into a nonrecombining region. Mating assays confirmed a sexual cycle in C. decagattii, demonstrating its ability to undergo mating and sporulation. Progeny analysis in K. mangrovensis revealed substantial ploidy variation and aneuploidy, likely stemming from haploid-diploid mating, yet evidence of recombination and loss of heterozygosity indicates that meiotic exchange occurs despite irregular chromosome segregation. Our findings underscore the importance of continued diversity sampling and provide further evidence for convergent evolution of fused MAT loci in basidiomycetes, offering new insights into the genetic and chromosomal changes driving reproductive transitions.

The family Ceratocystidaceae includes economically important plant pathogens that vary in host preference and lifestyle. These fungi are believed to attract insect vectors, for their dispersal through their floral and fruity scents. This study aimed to identify the volatiles produced by a subset of fungi within the Ceratocystidaceae using gas chromatography coupled with mass spectrometry. The primary volatiles produced by most genera in the family were fusel alcohols and fusel acetates, but their emission rates differed significantly between genera and isolates from a single species. Ceratocystis albifundus collected from Protea cynaroides produced higher levels of fusel acetates compared to isolates from Terminalia sericea. In addition, significant differences in volatile biosynthesis were observed between isolates grown under different temperatures. Results of this study demonstrate that Ceratocystidaceae exhibit varied volatile profiles, but further research is needed to understand the ecological and physiological mechanisms underlying this plasticity.

Fungi in dairy pastures impact cattle health, yet the diversity of fungal species present in South African pastures remains understudied. Following an outbreak of Sporidesmin-Induced Liver Disease (SILD; caused by the mycotoxin sporidesmin produced by Pseudopithomyces toxicarius) in the Eastern Cape in 2020, we collected mixed pasture samples from 14 dairy farms affected by this disease. Our aim was to investigate what fungal species are present in communities and whether species like Ps. toxicarius are present that may play a role in cattle health. A total of 708 strains were isolated from 95 mixed pasture samples and identified based on DNA sequence data to 132 species representing 55 genera. Fusarium was the most isolated (207 strains; 21 species; 55 samples), followed by Penicillium (75 strains; 22 species; 27 samples), Pseudopithomyces (69 strains; 2 species; 21 samples), Cladosporium (54 strains; 6 species; 23 samples), Epicoccum (52 strains; 6 species; 24 samples) and Bipolaris (38 strains; 3 species; 19 samples). Several strains could not be identified and represent potentially new or previously uncharacterised species. Additionally, phylogenetic analyses revealed the presence of Ps. palmicola and Ps. toxicarius in the Eastern Cape dairy pastures. Our findings underscore the ecological complexity of pasture environments and raise important questions about the role of fungal diversity in livestock health.

Organisms from all kingdoms of life release membrane vesicles, which are tiny, spherical structures made of a lipid bilayer. These vesicles carry out several functions, such as forming new cell membranes, removing waste products from the cell, and transporting lipids and other substances. The payloads often contained in the vesicles are sorted via the endosomal sorting complex required for transport (ESCRT) pathway stepwise. Furthermore, alterations to this endomembrane system reduces formation of vesicles and produce aberrant endosomal compartments. In pathogenic fungi, studies that have generated mutants with disruption in the ESCRT genes demonstrated negative effects on virulence and growth. Despite these important roles, only a few fungal species have to date been evaluated for the ESCRT pathway. In this review, we comprehensively evaluate recent developments in the ESCRT fungal pathway and its role in plant fungal pathogenesis.

The molecular mechanisms influencing Fusarium circinatum, an important pine tree pathogen, remain partially understood. We recently reported a biofilm-mediated response in this fungus, which supports its adaptation to harsh conditions including heat stress. Here, we report that biofilm extracellular vesicles (EVs) play a key role in this adaptive response. The EVs were purified from planktonic and biofilm cells via differential ultracentrifugation and size exclusion chromatography. Their subsequent application to fungal cells revealed the capacity of biofilm-derived EVs (bEVs) to promote conidial viability and germination. When bEV-treated conidia were cultured in the presence of carbon sources (PM1), nitrogen sources (PM3B) and chemical sensitivity agents (PM21D), a delayed nutrient utilisation and adaptation to antimicrobial agents such as nystatin were observed. Furthermore, exogenous application of bEVs on mono- and polymicrobial biofilms significantly enhanced biomass and matrix production, with EVs derived from heat-stressed biofilm (45 degrees C, 1 h) showing more effectiveness at promoting biomass production and resistance to the antifungal agent, tebuconazole. This is consistent with the biofilm's heat resistance previously reported for F. circinatum biofilms. Taken together, our work provides novel insights into the EV-mediated molecular interactions that modulate environmental responses in F. circinatum.

Cryphonectria canker is one of the most important diseases of plantation-grown Eucalyptus spp. in the tropics and Southern Hemisphere. The disease has been known in Florida, USA, for many years, and the causal agents are attributed to two known canker pathogens, Chrysoporthe cubensis (equivalent to Cryphonectria cubensis) and Microthia havanensis (equivalent to Endothia havanensis). These identifications were based on morphological characteristics, which are inadequate to recognise cryptic species in the Cryphonectriaceae. In this study, we visited various sites in Florida where Eucalyptus grandis and E. amplifolia trees are cultivated and investigated the presence of cankers. Isolations were made from fungal structures on symptomatic tissues associated with cankers. A total of 41 cultures resembling Cryphonectriaceae spp. were isolated. The isolates were identified based on DNA sequences for the ITS region of the rRNA and sections of the beta-tubulin gene, and confirmed as the three species of Cryphonectriaceae, namely Microthia havanensis, Chrysoporthe cubensis and Chrysoporthe doradensis. Of these, Chrysoporthe doradensis was discovered for the first time in the USA. In addition, this study represents the first record of any Cryphonectriaceae on E. amplifolia , a Eucalyptus species that is poorly studied in terms of disease. Pathogenicity trials on Eucalyptus showed that all three Cryphonectriaceae species could cause disease on these trees, with Chrysoporthe spp. being more aggressive. These findings highlight the importance of continuous monitoring and surveillance to detect emerging pathogens and safeguard the sustainability of Eucalyptus in non-native forestry systems.

Diplodia sapinea (Dothideomycetes) is a latent fungal pathogen with a global distribution that predominantly infects Pinus species. The impact of the fungus is increasing due to climate-driven range expansion and thus wide-scale disease outbreaks. With the aim of developing high quality genome resources, we generated chromosome-level genome assemblies for three D. sapinea isolates and low-coverage Illumina genome data for six additional isolates. By comparing these genome assemblies, we identified 14 core chromosomes and two accessory chromosomes (ACs) in the pathogen. The ACs encode 80 and 155 proteins, respectively, while 11374 - 11609 genes were identified in the core chromosomes. Both ACs had lower gene density and higher proportions of transposable elements compared to the core chromosomes. Sequence analysis indicated that genes on the ACs are rapidly evolving, suggesting they serve as evolutionary hotspots in the species. Sequence homology analyses suggested that the ACs were likely acquired horizontally, probably from a species in the Dothideomycetes. We designed PCR-based assays to aid in the detection of the ACs and applied these on a set of 37 isolates from 14 countries. One of the ACs was detected in 33 isolates from 13 countries, while the other AC was absent in all isolates tested. Pathogenicity trials on Pinus patula seedlings showed no correlation between the presence of ACs and isolate aggressiveness. The high-quality genomes provided here offer important resources for future research on this globally important pathogen, including the biological roles of the ACs.

This is a genome announacment there is no abstract

Quiver trees ( Aloidendron dichotomum ) are large iconic succulent plants found in arid areas of southern Africa. These trees have been observed suffering from die-back symptoms for many years. Various environmental and abiotic factors have been investigated as possible causes of the symptoms. However, biotic causes, especially microfungi that commonly cause die-backs in trees, have never been considered. During a routine disease survey, symptomatic stems and roots of the dying trees were collected in the Cape Province, South Africa. Isolations were made from tissues at the leading edges of the lesions on symptomatic stems and roots, and the resulting fungi were identified using morphological characteristics and DNA sequence data of four loci (LSU, SSU, ITS and β-tubulin). Five species were identified: Paecilomyces formosus , Phaeoacremonium ( Pm. ) parasiticum , Pm. luteum , Xylogone sphaerospora , and the newly described Coniophoma aloidendri gen. et sp. nov . Three species, Pm. parasiticum and C. aloidendri from this study and Alanphillipsia ( Ala. ) aloes were tested for their pathogenicity on A. dichotomum plants in a greenhouse trial. All three species gave rise to lesions significantly different in size from the controls. The Pm. parasiticum strains showed larger necrotic lesions than C. aloidendri and Ala. aloes . However, none of the isolated fungi were aggressive or are known as primary pathogens, and the cause of the die-back on symptomatic trees remains to be determined.

The ability to extract protoplasts has contributed significantly to the study of fungi and plants. Protoplasts have historically been used to determine chromosome number via pulsed-field electrophoresis and for the functional characterization of genes via protoplast transformation. More recently, protoplasts have been used to extract the high-molecular-weight DNA required for long-read sequencing projects. The availability of efficient protoplast extraction protocols is thus integral to the study and experimental manipulation of model and non-model fungi. One major hurdle to the development of such protocols has been the discontinuation of enzymes and enzyme cocktails used to digest the fungal cell wall. Here, we provide five protoplast extraction protocols for use in various filamentous ascomycete species spanning the genera Ceratocystis, Fusarium, Metarhizium, Ophiostoma, and Sclerotinia. These protocols all use an inexpensive, readily available enzyme cocktail called Extralyse, a commercially available product commonly used in the wine making industry. Using this enzyme cocktail overcomes reliance on the laboratory-grade enzymes that have frequently been discontinued and are often cost prohibitive at the concentrations required. The protocols described here will allow further research, including genome editing, to be conducted in these fungal genera. Importantly, these protocols also provide a starting point for the development of protoplast extraction techniques in other filamentous fungi. This resource can therefore be used to expand the molecular toolkits available for fungi beyond the species described here, including those with relevance in both medical and biotechnological industries. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Protoplast extractions from Ceratocystis eucalypticola and Ceratocystis fimbriata Basic Protocol 2: Protoplast extractions from Fusarium circinatum Basic Protocol 3: Protoplast extractions from Metarhizium acridum, Metarhizium brunneum, and Metarhizium guizhouense Basic Protocol 4: Protoplast extractions from Ophiostoma novo-ulmi Basic Protocol 5: Protoplast extractions from Sclerotinia sclerotiorum.

Fusarium circinatum, the causal agent of pine pitch canker, is one of the most destructive pathogens of Pinus species worldwide. Infections by this pathogen result in serious mortality of seedlings due to root and root collar disease, and growth reduction in trees due to canker formation and dieback. Although much is known about the population biology, genetics, and genomics of F. circinatum, relatively little is known regarding the molecular basis of pathogenicity in F. circinatum. In this study, a protoplast-based transformation using CRISPR-Cas9-mediated genome editing was utilized to functionally characterize a putative pathogenicity gene in three different strains of the fungus. In silico analyses suggested the gene likely encodes a small secreted protein, and all isolates in which it was deleted displayed significantly reduced vegetative growth and asexual spore production compared to the wild-type isolates. In pathogenicity tests, lesions induced by the deletion mutants on detached Pinus patula branches were significantly shorter than those produced by the wild-types. The putative pathogenicity gene was named Pgs reflecting its role in pathogenicity, growth, and sporulation. Future research will seek to explore the molecular mechanisms underlying the mutant phenotypes observed. Overall, this study represents a significant advance in F. circinatum research as the development and application of a Cas9-mediated gene deletion process opens new avenues for functional gene characterization underlying many of the pathogen's biological traits.

Biofilm formation was investigated in a maize rot-causing pathogen, Fusarium verticillioides. This work revealed that in vitro cultures produce structured, adherent communities with a dense extracellular matrix (ECM) surrounding hyphae that makes up the biomass of a matured biofilm. Pellicle containing exopolysaccharide had a hydrodynamic diameter of 4.19 nm and a low viscosity (0.022 dl/g). The exopolysaccharide was composed of amino sugars and unordered, facilitating stability through complexation with the anionic eDNA. Biofilm formation varied over different pH and temperature values, emphasising its role in promoting adaption, survival, and persistence in F. verticillioides, potentially contributing to its pathogenicity in maize. Collectively, the results provide valuable insights into biofilm structure and stress resistance in this fungus, and will serve as a foundation for future studies incorporating in planta infection systems.

A survey of the fungal diversity associated with mixed pastures from Eastern Cape dairy farms in South Africa led to the isolation of 155 Fusarium strains that belong to the Fusarium incarnatum-equiseti species complex (FIESC). Using single and multigene phylogenies based on partial sequences of the translation elongation factor 1-alpha (TEF), calmodulin (CaM), and the partial RNA polymerase second largest subunit (RPB2) genes, we identified 11 species. They included F. brevicaudatum, F. clavus, F. coffeatum, F. croceum, F. goeppertmayerae, and F. heslopiae, with five species that were found to be new. Based on morphological and phylogenetic data, three new species are formally described here as F. cumulatum, F. mariecurieae, and F. pascuum. We also provided a description for F. goeppertmayerae, as the authors who identified and named this species did not include one. We have chosen to not describe the remaining species, as our cultures lack proper morphological structure development. This study shows that mixed pastures harbour a diverse range of Fusarium species and highlights the need for further studies into their potential to impact animal health.