微生物学

Mycoparasitism is an important mechanism of fungal antagonism in which one fungus parasitizes another. This type of interaction plays a major role in the biocontrol activity of Trichoderma spp. against phytopathogenic fungi. Detailed visualization of these interactions is essential for understanding the structural mechanisms involved in fungal antagonism, including hyphal attachment, coiling, penetration, and cellular distortion. Scanning electron microscopy (SEM) is widely used for structural examination of fungal interactions; however, conventional preparation methods such as filter paper systems, membrane overlays, and agar block techniques often result in structural distortion, fragile sample handling, and difficulty in locating defined interaction zones. Here, we describe a modified slide-embedded technique for SEM visualization of mycoparasitic interactions between filamentous fungi. The protocol is adapted from previously reported slide culture approaches and involves embedding pre-cut sterile glass slide fragments directly into potato dextrose agar (PDA), followed by sequential inoculation of Fusarium sp. and Trichoderma viride. Fungal interactions occurring directly on the glass surface are subsequently subjected to fixation with 2.5% glutaraldehyde, graded ethanol dehydration, sputter coating, and SEM observation. Compared with conventional methods, the present approach provides improved handling stability, better preservation of native hyphal architecture, reduced deformation during processing, and easier localization of interaction zones during microscopy. The protocol also enables clear visualization of early antagonistic events such as hyphal coiling, penetration, and surface colonization. Due to its simplicity, reproducibility, and minimal technical complexity, this method serves as a practical and efficient approach for SEM-based investigation of fungal–fungal interactions and can be readily adapted for studying diverse mycoparasitic systems.

Vulvovaginal candidiasis (VVC), also known as vaginal thrush, is an infection of the vulvovaginal mucosa caused by fungi of the Candida genus. Particularly for patients suffering from recurrent infection, the disease has a significant impact on their quality of life. The still unknown aspects of disease pathogenesis, as well as factors driving the development of infections and recurrence, represent a challenge for both clinical practitioners and patients. Mouse models and patient studies have suggested important roles of the microbiome, deployment of fungal pathogenicity mechanisms in the vagina, and dysregulated immune responses for VVC pathology. Dissecting their individual contributions can reveal specific processes associated with infection and may inspire novel therapeutic strategies. Epithelial in vitro infection models have been playing a key role in dissecting a crucial interaction during VVC, the invasion and infection of the vaginal mucosa. They have been instrumental in characterizing candidalysin as a fungal toxin that damages epithelial cells and elicits initial inflammatory responses to catalyze downstream inflammation. Moreover, they have also revealed potential protective immune pathways. Such a standardized epithelial cell infection model offers high versatility and compatibility with different downstream assays to link epithelial responses with other processes during VVC. This protocol describes a general A-431 vulvovaginal epithelial cell–Candida infection model in detail and provides several adaptations, such as live-cell imaging and mRNA silencing, as well as possible follow-up readouts, like the quantification of cytokine release, cytotoxicity, and neutrophil recruitment to study diverse processes relevant to VVC research.

Sheath blight, caused by Rhizoctonia solani, is a major fungal disease of rice that leads to significant yield losses globally. Conventional inoculation methods often fail to achieve consistent and uniform infection, limiting their applicability in antifungal screening studies. This protocol describes a reliable in planta inoculation method for R. solani using mature sclerotia placed at the internodal region of tillering-stage rice seedlings. The procedure includes step-by-step instructions for seed germination, seedling preparation, pathogen culture, artificial inoculation, and post-infection application of antifungal treatments, including botanical compounds such as Ocimum gratissimum essential oil and thymol. Lesion development is monitored and quantified over time, and data are analyzed statistically to evaluate treatment efficacy. The protocol is optimized for reproducibility, scalability, and compatibility with sustainable disease management approaches. It provides a robust platform for evaluating antifungal agents in a biologically relevant and controlled environment.

It has been discovered that many phytopathogenic fungi can absorb exogenous double-stranded RNAs (dsRNAs) to silence target genes, inhibiting fungal growth and pathogenicity for plant protection. In our recent report, the beneficial arbuscular mycorrhizal (AM) fungi are capable of acquiring external naked dsRNAs; however, whether the dsRNAs can be delivered into AM fungi through nanocarriers remains to be investigated. Here, we introduce a simple and advanced method for in vitro synthesizing chitosan (CS)/dsRNA polyplex nanoparticles (PNs) to silence the target gene in the AM fungus Rhizophagus irregularis. This method is straightforward, requiring minimal modifications, and is both efficient and eco-friendly, offering potential for rapid application in elucidating gene functions in AM fungi.

The Fusarium genus includes various fungi of great significance in agriculture. Fusarium solani f. sp. eumartii (F. eumartii), traditionally known as a potato pathogen, has also been identified as a cause of disease in tomatoes. This protocol provides a detailed, efficient, and robust flood-inoculation method for assessing F. eumartii infection of young tomato seedlings grown on MS medium plates. It includes the evaluation of the lesion area and the quantification of the remaining fungal inoculum in tomato seedlings. In summary, the straightforwardness and efficiency of this bioassay make it a powerful quantitative tool for selecting fungicidal compounds or defense response inducers in tomato plants, offering a promising approach with significant potential for preventing fungal diseases in crops.

Most terrestrial plants are associated with symbiotic Glomeromycotina fungi, commonly known as arbuscular mycorrhizal (AM) fungi. AM fungi increase plant biomass in phosphate-depleted conditions by allocating mineral nutrients to the host; therefore, host roots actively exude various specialized metabolites and orchestrate symbiotic partners. The hyphal branching activity induced by strigolactones (SLs), a category of plant hormones, was previously discovered using an in vitro assay system. For this bioassay, AM fungi of the Gigaspora genus (Gigasporaeae) are commonly used due to their linear hyphal elongation and because the simple branching pattern is convenient for microscopic observation. However, many researchers have also used Glomeraceae fungi, such as Rhizophagus species, as the symbiotic partner of host plants, although they often exhibit a complex hyphal branching pattern. Here, we describe a method to produce and quantify the hyphal branches of the popular model AM fungus Rhizophagus irregularis. In this system, R. irregularis spores are sandwiched between gels, and chemicals of interest are diffused from the surface of the gel to the germinating spores. This method enables the positive effect of a synthetic SL on R. irregularis hyphal branching to be reproduced. This method could thus be useful to quantify the physiological effects of synthesized chemicals or plant-derived specialized metabolites on R. irregularis.

Magnaporthe oryzae is a filamentous fungus responsible for the detrimental rice blast disease afflicting rice crops worldwide. For years, M. oryzae has served as an excellent model organism to study plant pathogen interactions due to its sequenced genome, its amenability to functional genetics, and its capacity to be tracked in laboratory settings. As such, techniques to genetically manipulate M. oryzae for gene deletion range from genome editing via CRISPR-Cas9 to gene replacement through homologous recombination. This protocol focuses on detailing how to perform gene replacement in the model organism, M. oryzae, through a split marker method. This technique relies on replacing the open reading frame of a gene of interest with a gene conferring resistance to a specific selectable chemical, disrupting the transcription of the gene of interest and generating a knockout mutant M. oryzae strain.

Key features

• Comprehensive overview of primer design, PEG-mediated protoplast transformation, and fungal DNA extraction for screening.

Barley (Hordeum vulgare) is one of the most important agricultural crops in the world, but pathogen infections regularly limit its annual yield. A major threat is the infection with the biotrophic leaf rust fungus, Puccinia hordei. Rust fungi have a complex life cycle, and existing resistances can be easily overcome. To address this problem, it is crucial to develop barley varieties with improved and durable resistance mechanisms. An essential step towards this goal is a simple and reproducible infection protocol to evaluate potential resistance phenotypes in the lab. However, available protocols sometimes lack detailed procedure or equipment information, use spore application methods that are not suitable for uniform spore dispersion, or require special mineral oils or engineered fluids. In addition, they are often optimized for pathogen-dedicated greenhouses or phytochambers, which may not be available to every research institute. Here, we describe an easy and user-friendly procedure to infect barley with Puccinia hordei on a small laboratory scale. This procedure utilizes inexpensive and simple tools to evenly split and apply spores to barley leaves. The treated plants are incubated in affordable and small phytocabinets. Our protocol enables a quick and reproducible infection of barley with leaf rust, a method that can easily be transferred to other rust fungi, including stripe rust, or to other plant species.

Key features

• Step-by-step infection protocol established for barley cv. Golden Promise, the gold standard genotype for genetic transformation

• Plant age–independent protocol

• Precise spore application by using inexpensive pipe cleaners for uniform symptom formation and increased reproducibility

• No specialized equipment needed

• Includes simple spore harvesting method

• Protocol is applicable to other biotrophic pathogens (stripe rust or powdery mildew) and other plants (e.g., wheat)

• Protocol is also applicable for a detached leaf assay

Graphical overview

Cryptococcus neoformans is a human pathogenic fungus that can cause pulmonary infections and meningitis in both immunocompromised and otherwise healthy individuals. Limited treatment options and a high mortality rate underlie the necessity for extensive research of the virulence of C. neoformans. Here we describe a detailed protocol for using the Galleria mellonella (Greater Wax Moth) larvae as a model organism for the virulence analysis of the cryptococcal infections. This protocol describes in detail the evaluation of G. mellonella larvae viability and the alternatives for troubleshooting the infection procedure. This protocol can be easily modified to study different inocula or fungal species, or the effects of a drug or antifungal agent on fungal disease within the larvae. We describe modified alternative versions of the protocol that allow using G. mellonella to study fungal diseases with different inocula and at different temperatures.

Rhizoctonia solani is a soil-borne fungus, which rarely produces any spores in culture. Hence, all inoculation procedures are based on mycelia, often as a coat on cereal kernels, placed in close vicinity to the plant to be infected. In this protocol, an inoculation method is described where the fungus is first allowed to infest a perlite-maize flour substrate for 10 days, followed by thorough soil mixing to generate uniform fungal distribution. Pre-grown seedlings are then replanted in the infested soil. Plant materials can be harvested, five (sugar beet) and ten days (Arabidopsis) post infection, followed by a rapid cleaning step ahead of any nucleic acid preparation. Commercial DNA or RNA extraction kits can be used or, if higher DNA yield is required, a CTAB extraction method. Our purpose was to develop a reliable and reproducible protocol to determine the infection levels in planta upon infection with R. solani. This protocol is less laborious compared to previous ones, improves the consistency of plant infection, reproducibility between experiments, and suits both a root crop and Arabidopsis.

Graphic abstract:

Overview of the R. solani infection procedure.