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.

The transcription factor Pho4 is crucial for the response to phosphate starvation in many fungi, and it has been linked to tolerance to alkalinization of the medium and to pathogenicity. It is widely accepted that it is encoded by a single gene. However, the industrially relevant yeast Komagataella phaffii might contain two Pho4-encoding genes (PAS_chr1-1_0265 and PAS_chr2-1_0177, designated here PHO4(A) and PHO4(B), respectively), which have never been functionally characterized. The phenotypic analysis of single and double mutants suggests that Pho4(B) plays a major role in the adaptation to Pi scarcity. While single mutants exhibited limited and non-overlapping phenotypic defects, the pho4(A) pho4(B) strain was sensitive to multiple types of stress, including phosphate starvation and alkaline pH. Transcriptomic analysis confirms that Pho4(B) is crucial for the transcriptional response to phosphate starvation, including induction of typical gene markers (PHO5, PHO89, VTC1, etc.). However, by using a GFP reporter we found that PHO4(A) also participates in the induction of PHO89 under high pH stress. Expression of both PHO4(A) and PHO4(B) in S. cerevisiae complemented the pho4 mutation under phosphate limitation by restoring growth, expression of the Pho84 transporter and secreted phosphatase activity. These results indicate that both transcription factors display partially overlapping functions, responding differently to diverse stimuli, and that together they constitute a key component in the adaptation to a variety of stresses. Therefore, K. phaffii is an exceptional example among fungi that encodes two Pho4 functional transcription factors.

AbstractEmergomyces africanus is a thermally dimorphic fungal pathogen endemic to Southern Africa which can cause fatal systemic infections in persons with advanced HIV disease. Its mechanisms of pathogenesis are not well understood. Characterisation of virulence traits in this pathogen requires appropriate molecular tools for genetic manipulation. Molecular technologies developed for the transformation of H. capsulatum were adapted for use in E. africanus. Agrobacterium-mediated transformation was used to generate a reporter strain expressing green fluorescent protein (GFP). The E. africanus GFP reporter strain facilitated the study of yeast interaction with macrophages in vitro and allowed the identification of infected phagocyte cell types in the mouse lung by flow cytometry. E. africanus could also maintain episomal plasmids with telomere-like sequences, to introduce expression constructs without genome modification. Using this plasmid system, RNA interference constructs were used to knock down the expression of cell wall (1,3)-glucan by targeting the transcripts of the -glucan synthase (AGS1). An episomal CRISPR/Cas9 system was evaluated for E. africanus, which effectively disrupted GFP in a reporter strain and enabled the generation of a URA5 uracil auxotroph. These tools and strains will facilitate future studies to elucidate the mechanisms of pathogenesis of E. africanus. ImportanceEmergomyces africanus is an opportunistic fungal pathogen affecting persons with advanced HIV disease in South Africa. The biology and pathogenesis of E. africanus are not well understood, as the importance of the disease caused by this fungus (emergomycosis) has only been recognised in recent years and molecular studies have been impaired by the lack of genetic technologies. In this work, we describe tools and methods for the genetic modification of this pathogen, which will accelerate future studies investigating how the fungus causes disease in the human host. These essential tools include (1) the ability to create fluorescent reporter strains, such as the green fluorescent protein E. africanus strain described here, which facilitates tracking the spread of the fungus during infection and enhances microscopy studies, (2) methods for knocking down gene expression in E. africanus, and (3) the permanent disruption of genes through CRISPR/Cas9 gene editing.

BackgroundThe environment in which a fungus grows can directly influence their development, transmission, and pathogenic potential. This environment encompasses factors like nutrient availability, biotic and abiotic stressors, as well as host-derived chemical cues. In fungal pathogens, where conidia act as the infectious agents, the environment impacts the quantity and quality of these spores, thereby aOecting their ability to infect and kill hosts. In the present study, we investigated the effect of host-derived medium types on various phenotypes, including spore production, growth rate, and virulence in two entomopathogenic fungi, Metarhizium acridum and Metarhizium brunneum. Three medium types derived from insect material were compared to a standard laboratory medium. ResultsConidia produced on the insect-derived media exhibited enhanced sporulation and reduced time to sporulation, while conidial germination and maximum growth rate were comparable across medium types, suggesting that some of the medium-induced phenotypic effects were transient. Notably, conidia derived from two of the insect medium types demonstrated higher virulence, indicating that host-derived cues may prime virulence. ConclusionThese results highlight that the composition of growth substrates can regulate fungal reproductive strategies and virulence, with implications for developing high-throughput phenotyping and for the biotechnological optimization of mass production and efficacy of entomopathogenic fungi in biological control applications. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/711814v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@189013eorg.highwire.dtl.DTLVardef@1b0cedborg.highwire.dtl.DTLVardef@dccb4eorg.highwire.dtl.DTLVardef@1a77895_HPS_FORMAT_FIGEXP M_FIG C_FIG

The auxin-inducible degron (AID) technology is a convenient and powerful tool for protein functional characterization in a broad array of eukaryotic species. We recently demonstrated that the original AID and improved AID2 systems are very effective at rapid protein depletion in Candida albicans and described a limited set of reagents for their use in certain auxotrophic lab strains. With an eye towards broader applicability with improved flexibility, we report here a new series of template vectors suitable for employing AID2 technology in prototrophic C. albicans strains, including clinical isolates. We adapted a common recyclable antibiotic marker system for the required genome editing steps and developed a strategy for simultaneous CRISPR/Cas9-mediated tagging of both target alleles. We also developed a composite all-in-one tagging cassette that combines the degron tag and the OsTIR1F74A gene for single step strain engineering. We added a fluorescent protein tag option and designed and validated an approach for N-terminal tagging that retains natural promoter control. We also compared effectiveness of the two commonly used synthetic auxins, 5-Ph-IAA and 5-Ad-IAA and the two common OsTIR1 variants, F74A and F74G, and provide guidelines for using the new AID2 system. Finally, using the novel all-in-one cassette, we demonstrate that the AID2 system also works in Candida auris. The new reagents should enhance the convenience and accessibility of the AID2 system for the Candida research community. IMPORTANCEInvasive fungal infections, including those caused by Candida species, are a persistent global health problem, and their treatment is hindered by limited antifungal options and the emergence of drug resistance. There is an urgent need for tools and methods to accelerate discovery of novel therapeutic targets. The expanded and optimized auxin-inducible degron system described herein provides a versatile platform for characterizing protein function and dissecting pathways governing important traits like virulence, stress tolerance, and antifungal resistance. The new reagents make AID technology applicable to any strain. Ultimately, this enhanced toolkit has the potential to help identify and validate new high-value drug targets and deepen our understanding of molecular mechanisms that drive pathogenicity of Candida and other fungal pathogen species.

Histoplasma spp. is a dimorphic fungal primary pathogen that infects people worldwide and frequently affects immunosuppressed patients. Previous studies have identified the AMY1 gene product, the -amylase Amy1p, as essential for -glucan production and virulence in Histoplasma capsulatum. We identified two new genes (AMY2 and AMY3) in the Histoplasma genome that encode putative -amylases and made mutants using CRISPR/Cas9 technology, followed by evaluation of their role in -glucan biosynthesis and virulence. We also searched for AMY gene copies in 19 fungal genomes with the goals of identifying orthologs for AMY2 and AMY3, and establishing how many AMY copies existed across different fungi. We found that the number and type of -amylases vary depending on the fungal species; that all -amylases related to Histoplasma Amy1p belong to the GH13_5 subfamily, and all orthologs related to Histoplasmas Amy2p and Amy3p belong to the GH13_1 subfamily. We performed phylogenetic analyses of the three paralogs and revealed that the Histoplasma AMY duplications are ancient. We further established Amy2 is an ortholog of Aspergillus niger AgtA, and Aspergillus nidulans AmyD, and that it is partially involved in Histoplasma -glucan biosynthesis and virulence, while Amy3p is an ortholog of Aspergillus flavus Amy1, and it is dispensable for -glucan biosynthesis and virulence.

The fastidious basidiomycete Rhizoctonia (Ceratobasidium) theobromae is a biotrophic pathogen that causes Vascular-Streak Dieback (VSD) of Theobroma cacao (cocoa). The fungus has also been identified as the cause of an emergent disease known as Cassava Witches Broom Disease (CWBD) raising concerns that the pathogen is spreading to alternative hosts and to new regions. Interestingly, while VSD of cocoa and CWBD are reported as co-present in several countries, there is currently no evidence for cross-infection between species. The fungus is difficult to culture in vitro due its slow growth and Kochs postulates have not been definitive on either host. The complete fungus life cycle therefore remains enigmatic, though studies have progressed knowledge on pathology within the both the cocoa and cassava hosts. We have conducted limited field trials and sequenced mating (MAT) and ITS loci of isolates from various infected hosts and regions. We hypothesize that (i) genetic variation at MAT loci correlates with region or host (ii) long amplicon ITS sequences between isolates are more definitive for polymorphisms (iii) life-cycle traits of R. theobromae may be inferred from MAT loci (iv) cassava grown under VSD infected cocoa will be infected and develop symptoms of CWBD. We did not find any cross-infection in field trials, and we show that the pathogen is highly homozygous, despite undergoing meiosis, indicating a predominantly homothallic life cycle. Our data indicate that the pathogen is likely host specific and regionally divergent and suggests that host specificity on cocoa and cassava evolved by selection from a common ancestor rather than a host jump.

Filamentous fungi, such as Aspergillus species, use microtubule transport to move early endosomes. Other cargos, such as peroxisomes and mRNAs, "hitchhike" on early endosomes to move throughout the long hyphae of these organisms. In Aspergillus nidulans, peroxisomes hitchhike on early endosomes using the endosomal protein PxdA and the peroxisomal protein AcbdA. The HookA adaptor protein links endosomes to microtubule motors. Here, we set out to explore the physiological functions of peroxisome hitchhiking and endosome motility. A. nidulans has a complex life cycle that includes asexual and sexual reproduction. A. nidulans and other fungi within the Pezizomycotina subphylum are also notable for the vast number of secondary metabolites they produce. Light and other environmental conditions influence developmental decisions and secondary metabolite production. Here, we found that sexual reproduction is favored in the absence of endosome motility, even in the light, which normally promotes asexual reproduction. RNA sequencing of strains lacking endosome motility showed altered expression of genes involved in development. Unexpectedly, we observed altered expression of genes involved in secondary metabolism in strains lacking endosome motility and peroxisome hitchhiking. Using mass spectrometry, we found that the loss of endosome motility affected the biosynthesis of secondary metabolites, including sterigmatocystin, a carcinogenic mycotoxin that is a food contaminant. Finally, in a pathogenic species, Aspergillus fumigatus, we found that deletion of its PxdA homolog also significantly altered secondary metabolite production. Our work uncovers an unexpected link between organelle motility, developmental decisions in response to light, and secondary metabolite production in filamentous fungi.

The objective of this study was to re-analyse the molecular phylogeny and/or the morphology of all species, which have been attributed to the so-far mono-generic fungal family Ambisporaceae. The genus Ambispora has been well-known for its spore bi-morphy described even from single spore clusters. Triple-walled spores are differentiated on sporiferous saccules, while mono-walled spores are formed on simple subtending hyphae. New phylogenetic analyses reveal dissimilarities of [≥]10% in partial nrDNA gene of three different stable phylogenetic clades and thus suggest the division of Ambispora into three genera, which simultaneously request for advanced morphological separations. These advances are primarily based on the more diverse spore wall composition of the ambisporoid-acaulosporoid morph rather than on the rather simple-glomoid morph. While all known species of the triple-walled morph have an evanescent to semi-permanent outer spore wall, i) Am. fennica, Am. brasiliensis, Am. gerdemannii and Am. nicolsonii have a smooth, permanent central spore wall (Am. fennica clade, A), ii) the central wall of Am. appendicula, Am. callosa, Am. leptoticha and Am. jimgerdemannii is alveolate (Am. appendicula clade, B), and iii) the central wall of Am. granatensis is smooth, but easily degraded, thus rather short-lived and not permanent but evanescent (Am. granatensis clade, C). In conclusion, species of the Am. fennica clade represent the genus Ambispora, while species of the Am. appendicula clade represent the new genus Appendiculaspora, and the mono-specific Am. granatensis clade represents the new genus Ephemerapareta. Species of an additional morph, with triple-walled spores, but apparently formed on subtending hyphae, and having a diagnostic reticulate, football-like middle wall, are here separated from the revised genus Ambispora based solely on morphological analyses, since molecular identification analyses so far failed and remained merely unknown. This later morph and genus is based on the type species Pelotaspora reticulata comb. nov, and on P. austrolatina sp. nov. Concomitant molecular phylogenetic and morphological analyses are needed to attribute not only Pelotaspora spp., but also those species, for which hitherto only the ambisporoid-glomoid morph has been observed correctly within the family Ambisporaceae. Without molecular analyses, such species with glomoid but unknown ambisporoid-acaulosporoid morph have to be retained within Ambispora.

Nakaseomyces glabratus is a globally distributed opportunistic fungal pathogen. An ongoing discussion in studies of N. glabratus population structure has been whether genetic clusters are best defined using multilocus sequence typing (MLST) or short-read whole-genome sequencing (WGS). To assess the concordance between MLST- and WGS-based phylogenies, we analyzed a dataset of 548 N. glabratus WGS sequences from 12 countries. Clusters identified from WGS largely recapitulated the MLST-defined sequence type (ST) groups: fourteen WGS clusters were composed of a single MLST ST, and the remaining contained STs with very closely related MLST profiles. We thus propose a pragmatic naming convention, consistent with the system used in other microbial species, which specifies WGS cluster labels based on the primary ST. From the large WGS isolate dataset, we determined the prevalence of admixture and genomic variants. Interestingly, seven of the nine singleton isolates were admixed, in addition to 58 isolates from six different clusters. Aneuploidy was detected in 4% of isolates, most commonly in chrE, which contains ERG11, the gene encoding the enzyme targeted by azole antifungals. Aneuploid chromosomes did not exhibit elevated heterozygosity relative to the sequencing error rate, consistent with instability of extra chromosome copies. Copy number variants were found in 3% of the isolates; some of the CNVs co-occurred with aneuploidies, and were primarily identified on chrD, chrE, chrI, and chrM. Our findings demonstrate that deep splits between clusters preserve the utility of MLST ST designations for clade-level designation, yet underscore the utility of WGS for high-resolution genomic analyses. Article SummaryThere is an ongoing debate in studies on Nakaseomyces glabratus about whether traditional MLST analysis is sufficient to determine population structure, or whether the precision of whole genome sequencing (WGS) is necessary. We analyzed WGS data from 548 isolates from around the world. We found a very strong agreement between the two methods. We propose a hybrid naming system, where cluster names are based on the dominant MLST group. We used the WGS data to show that admixed isolates, and those with extra chromosomes or CNVs are rare (<7% of isolates in each class) and are distributed throughout the phylogeny.

Currently, the fungal class Archaeosporomycetes consists of one order, Archaeosporales with four families: Archaeosporaceae, Ambisporaceae, Geosiphonaceae, and Polonosporaceae. In the present study, the objective was to re-analyze the phylogeny and morphology of the Archaeosporomycetes from order to genus level. The different ecological strategies and, consequently, distinct evolutionary patterns of these taxa, as well as their morphological characters and other data updated here, suggest the need to divide Archaeosporales into four orders: (i) the type order Archaeosporales, (ii) Ambisporales ord. nov., both with four genera, (iii) Geosiphonales and (iv) Polonosporales ord. nov., both with single families and genera. Remarkably, the order Geosiphonales was described in the past, but was not considered in the Archaeosporomycetes until now. Phylogenetically, the four main clades (orders here proposed) of Archaeosporomycetes are well supported, with bootstrap values higher than 95% in all analyses, except Ambisporales/Ambisporaceae for RAxML-NG FBP analysis in the SSU tree (75%). Ecologically, this class includes three orders of arbuscular mycorrhizal fungi (AMF) forming symbiotic associations with plants, while Geosiphonales form an endocytobiosis with the cyanobacterium Nostoc. Morphologically, there are at least two AMF orders with spore bimorphism, which has not (yet) been described for Polonosporales. The only known species of Polonosporales, Polonospora polonica, forms spores directly on the neck of sporiferous saccules and the spores can morphologically be differentiated from all other taxa in Archaeosporomycetes by the formation of three permanent, rather thick spore walls, of which two form de novo during spore formation. The outer spore wall of Archaeosporales and Ambisporales are semi-permanent, evanescent or even short-lived, or show multiple fissures during aging, when it is more resistant. Ambisporales can easily be differentiated from Archaeosporales for instance by larger spores of the acaulosporoid morph and thicker spore walls. Our phylogenetic analyses suggested that Archaeosporales can be divided into two families: Antiquisporaceae that was described to form intraradical hyphae, vesicles and spores, staining darkly in Trypan blue, and Archaeosporaceae whose hyphae generally do not or only faintly stain in this reagent, and vesicles and intraradical spores have been rarely, if ever reported.

Candida auris (Candidozyma auris) is an emerging multidrug-resistant fungal pathogen that poses a significant global health threat. However, the molecular mechanisms underlying its virulence remain incompletely understood. In this study, we performed in vivo transcriptome analysis using an immunosuppressed mouse gastrointestinal infection model to identify genes associated with host-adaptation and virulence during infection. By comparing fungal transcriptomes obtained from colonization and dissemination sites with those from in vitro cultures, we identified genes that were consistently upregulated during infection. Among these genes, the unfolded protein response regulator HAC1 was selected as a candidate virulence-associated gene for further analysis. RT-PCR and sequencing analyses revealed that HAC1 mRNA in C. auris undergoes an unconventional splicing event of 287 bp that is enhanced under ER stress conditions. The excised region spans the annotated open reading frame boundary, suggesting that the translated region of HAC1 may require re-evaluation. Notably, a proportion of HAC1 transcripts appeared to be spliced even under non-stress conditions, indicating a detectable basal level of UPR activation. Differences in splicing dynamics were also observed among clade strains. Functional analyses demonstrated that deletion of HAC1 increased sensitivity to ER stress and heat stress. The HAC1 deletion mutant also exhibited reduced virulence in both Galleria mellonella and immunosuppressed mouse infection models, as evidenced by delayed host mortality and decreased fungal burdens, respectively. These findings indicate that HAC1 contributes to ER stress adaptation, thermotolerance, and survival in the host environment, and identify HAC1 as a virulence-associated gene in C. auris.

Aspergillus fumigatus is a world-wide saprophyte filamentous fungus which released conidia, its infectious morphotype, in the atmosphere. These conidia are inhaled daily by humans and can colonize the respiratory tract, where they may develop into hyphae, the invasive morphotype. We previously showed that bronchial epithelial cells (BECs) restrict A. fumigatus virulence by inhibiting conidial germination and filament formation through a process requiring PI3K signaling and the conidial fucose-specific lectin FleA. In the present study, we are looking to identify host factors and cellular partners involved in the BEC antifungal response and to define the molecular interactions underpinning FleA recognition. For this, we analyzed transcriptome of BECs infected with A. fumigatus in the presence or absence of the PI3K inhibitor LY294002. Functional involvement of candidate genes was assessed by siRNA knockdown and readouts of fungal filamentation (microscopic scoring and galactomannan release). FleA-interacting host proteins were identified by biotin-FleA affinity co-precipitation coupled to Tandem mass spectrometry, and validated by surface plasmon resonance and biolayer interferometry. The spatiotemporal dynamics of FleA and candidate partners were analyzed by confocal microscopy and proximity ligation assay We demonstrated that BEC antifungal activity involves at least two complementary pathways: a PI3K/laminin-332 axis promoting conidial adhesion, and a FleA-dependent pathway engaging ITGB1 and MRC2 consistent with lectin uptake and trafficking toward LAMP1-positive compartments. These findings nominate FleA-host receptor interactions as attractive targets for anti-adhesive strategies against A. fumigatus. Author summaryFungal pathogens are an increasing threat to public health, as they are becoming more common and harder to treat due to rising drug resistance. Among them, Aspergillus fumigatus has been classified as a critical pathogen by the World Health Organization (WHO). This filamentous fungus delivers spores in the air daily, which are constantly inhaled by humans. In people with weakened immunity, these spores can cause a range of lung diseases known as aspergillosis, with severity ranging from mild to life-threatening. Lung epithelial cells are the first cells of the respiratory tract to encounter inhaled spores. In a previous study, we showed that bronchial cells can prevent spore from developing into filaments, the invasive form of A. fumigatus that is responsible for tissue damage. This protective effect depends of on the recognition of a fungal protein called FleA. In the present study, we identified host cell proteins that bind to FleA and transport it into intracellular compartments. Our findings suggest that these proteins help bronchial epithelial cells to internalize fungal spores, thereby blocking their transformation into the invasive filamentous form.

Banana leaf diseases are a significant threat to Cavendish banana production. In the Philippines, the main disease has been diagnosed as Black sigatoka disease caused by Pseudocercospora fijiensis based on symptoms. However, our study showed that the main pathogen in Mindanao island, the largest banana-producing region in the Philippines, belongs to the genus Nigrospora, contradicting previous assumptions. We clarified the phylogenetic positions of 160 Nigrospora isolates based on molecular phylogenetic analyses using ITS, {beta}-tubulin, and tef1 sequences, and compared their morphology with known species. Molecular phylogenetic and morphological analysis revealed that Nigrospora isolates comprised N. chinensis, N. lacticolonia, N. cf. singularis, N. sphaerica, N. vesicularifera, and a novel species, N. nigrocolonia. Pathogenicity tests on banana leaves confirmed that these species are pathogenic. Species other than N. sphaerica were for the first time reported as pathogens of banana leaf. The results of the fungicide sensitivity test using 14 fungicides, including pyrimethanil, spiroxamine, and tebuconazole, for the Sigatoka disease showed 100% inhibition of all isolates at 100 ppm of active ingredients. However, low-sensitivity isolates were observed for the remaining 11 fungicides. Our findings indicated the need for a comprehensive review of banana leaf disease prevention strategies.

Leaf diseases pose a serious threat to faba bean production. Leaf blotch of faba bean, caused by Alternaria spp., has become increasingly widespread and destructive in several countries. Leaf diseases pose a serious threat to faba bean production. The infection of plant by pathogens can be influenced by various factors associated with the host plant, environmental conditions and presence of other microorganisms. The phyllosphere and endosphere play a critical role in plant health and disease development. This study aimed to evaluate the factors shaping the structure and diversity of fungal communities associated with faba beans. Plant samples were collected in 2004 from two intensively managed faba bean production fields in the central region of Latvia. Fungal assemblages were characterized using an ITS region metabarcoding approach based on Illumina MiSeq sequencing. Among the assigned amplicon sequence variant (AVS), 65% belonged to the phylum Ascomycota, while approximately 4% were classified as Basidiomycota. Alternaria and Cladosporium were the dominant genera across samples. The alfa and beta diversities of fungal communities was higher during flowering of faba beans to compare with ripening. The higher abundance of Basidiomycota yeasts were observed during flowering, in contrast, Cladosporium genus was significantly more abundant during ripening. Alternaria DNA was found on leaves that showed no symptoms of the disease. The diversity and composition of fungal communities were significantly influenced by sampling time and presence of leaf blotch, caused by Alternaria spp.

Fusarium wilt of banana, caused by Fusarium oxysporum f. sp. cubense race TR4 (Foc), is one of the most destructive diseases threatening global banana production, particularly the Cavendish cultivar. Conventional control strategies, including chemical treatments and quarantine, remain largely ineffective and unsustainable, underscoring the urgent need for alternative approaches. Biological control using rhizosphere-associated microorganisms offers a promising and environmentally friendly strategy. In this study, we isolated 436 bacterial strains from the rhizosphere of healthy banana plants and screened them for antifungal activity against Foc. Out of the screened isolates, 93 exhibited significant in-vitro inhibitions, and 64 of these were subsequently evaluated in greenhouse assays. We found that 22 strains reduced Fusarium wilt severity by 45-85% compared to untreated controls. Among them, two isolates, DDC20 and DDC_NEW2, consistently demonstrated strong biocontrol activity. In addition, cell-free culture media (CFCM) and crude extracts inhibited spore germination in fluorescence-based assays, indicating the involvement of secreted antifungal metabolites. Microscopy and confocal observations of GFP-tagged Foc revealed hyphal abnormalities in the presence of bacterial treatments, including swelling, irregular branching, and distortion, accompanied by excessive sporulation characterized by abundant microconidia, macroconidia, and chlamydospores. Whole-genome sequencing and comparative analyses placed both isolates within the genus Bacillus. Genome mining using antiSMASH identified multiple biosynthetic gene clusters encoding known antifungal compounds such as surfactin, fengycin, bacillibactin, and difficidin, as well as putative novel clusters. LC-MS confirmed the presence of surfactin and fengycin in bacterial extracts, supporting the genomic predictions. Collectively, these findings highlight the potential of DDC20 and DDC_NEW2 (related to Bacillus spp.) from the banana rhizosphere as effective biocontrol agents against Foc TR4. This integrated approach, combining phenotypic assays, microscopy, and genome mining, provides a strong foundation for the development of sustainable strategies to manage Fusarium wilt in banana cultivation.

During host-pathogen interactions, fungal pathogens secrete effector proteins into host cells to manipulate the host immune system and facilitate infection. Although many effector genes are highly expressed during infection stages, there is limited information on the mechanisms regulating their in planta expression. Here, we characterize the promoter of MoHTR1, a nuclear effector gene of the rice blast fungal pathogen, to elucidate its in planta-specific expression. Using promoter deletion and mutation analyses, we identified a core cis-element (TATTTCGT) within the MoHTR1 promoter, designated the in planta active (IPA) element, which is crucial for in planta-specific expression. The IPA element is responsible for the expression of not only MoHTR1, but also other effector genes including a known effector Slp1. Furthermore, the IPA element enables the in planta expression of MobZIP14, a gene specifically expressed during vegetative growth. The IPA element plays a critical role in fungal virulence by enabling MoHTR1 expression and regulating host immune responses. Bioinformatic and DNA-protein interaction analyses revealed that RGS1, a transcription factor containing a winged-helix binding domain, acts as a transcriptional regulator of MoHTR1 by directly binding to the IPA element. Our findings provide new insights into the regulatory mechanisms driving the in planta-specific expression of fungal effector genes.

Filamentous fungi have complex, three-dimensional growth patterns and a non-adherent nature, which can present challenges for live-cell imaging for quantitative assessment of dynamic cellular processes. To address these challenges, a live-cell imaging system has been modified to constrain the model fungus Aspergillus nidulans to growth in a single focal plane. This enables high-resolution time-lapse imaging of actin dynamics throughout development using a Lifeact actin marker. This system was used to perform kymographic analysis to quantify actin velocity and hyphal extension rates during early hyphal development. Results show two distinct growth phases: germ tube extension (0.58 m/min) and hyphal extension (1.52 m/min). Actin exhibited bi-directional transport along hyphae with biased movement toward the spore body. Actin was also observed re-localizing from hyphal tips to sites of septum formation indicating active redistribution of cytoskeletal resources based on cellular demands. This technological advancement overcomes longstanding limitations in fungal live-cell imaging and provides a new platform for quantitative systems-level analysis of mycelial development, offering new insights into the spatiotemporal coordination of cytoskeletal dynamics during filamentous growth.

Human colonization since the 19th century has resulted in the global spread of pines across the Southern Hemisphere, well beyond their original northern boreal distribution. Such introductions moved not only the pines but also expanded the distribution of their symbiotic partners. Although the introduction of pines is documented through historical records, little is known about the introduction history of their ectomycorrhizal fungi, which are critical symbionts for the survival and invasion of pines. Using Suillus luteus as an example, population genomic analyses of 208 individuals across both native and introduced ranges showed that all introductions originated from Europe, likely mediated by human activities along with pine introductions. With the exception of North America, introduced populations were genetically differentiated from the Europe population, with varying magnitudes of population expansion in different introduced regions, often linked to forestry practices. Genetic variation within the native European population followed isolation by distance, but not in the introduced range, highlighting the disparity in the spatial genetic patterns of native versus exotic habitats. This study provides insight into the population genetics of a globally introduced ectomycorrhizal fungus whose introduction process is likely applicable to other pine-co-introduced ectomycorrhizal fungi.

Australia is the largest producer of Pyrethrum (Tanacetum cinerariifolium) globally. Amongst the constraints on production are the fungal pathogens Didymella tanaceti and Stagonosporopsis tanaceti, which pose a significant threat to the industry, causing substantial yield losses. While the infection biology of S. tanaceti is well characterised, knowledge of D. tanaceti and its potential interaction with S. tanaceti on plants remains limited, hindering disease management. We developed fluorescently labelled strains of both pathogens via Agrobacterium tumefaciens-mediated transformation (ATMT). Binary vectors carrying the mNeonGreen or tdTomato fluorescent protein genes were introduced into D. tanaceti and S. tanaceti, respectively, and expression of the fluorescent proteins was confirmed by microscopy. Genome sequencing revealed single-copy T-DNA insertions in all transformants, with minor genomic rearrangements at insertion sites. Detached leaf assays demonstrated that transformed strains retained pathogenicity, producing disease symptoms indistinguishable from those of the wild type. These fluorescently labelled variants enabled detailed visualisation of D. tanaceti infection biology and its interactions with S. tanaceti, including co-infection dynamics. Co-infection assays using fluorescent strains further facilitated simultaneous visualisation and differentiation of both pathogens within host tissues. Importantly, these tools also allowed the first description of the early stages of infection by D. tanaceti in pyrethrum leaves. This study represents the first successful transformation of D. tanaceti and S. tanaceti, providing valuable resources to investigate their infection processes.