Microbiology

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.

Identifying microscopic mycorrhizal fungal structures in roots, i.e., hyphae, vesicles and arbuscules, requires root staining procedures that are often time consuming and involves chemicals known to present health risks from exposure. By modifying established protocols, our root staining method stains roots using a safe ink- and vinegar-based staining solution, followed by a 2-16 h-long de-staining period. The entire procedure can be completed in less than 6 h (plus up to 16 h de-staining overnight) and roots are suitable for semi-permanent and permanent slide mounting for light microscopy. We tested our method on hundreds of wild-sourced roots from two different plant species: Lycopodiella inundata, a herbaceous clubmoss with tough water-resistant roots, and Sambucus nigra, a temperate woody shrub. Both plants associate with endomycorrhizae, L. inundata predominantly with Mucoromycotina fine root endophytes (MucFRE) and S. nigra with Glomeromycota arbuscular mycorrhizal fungi (AMF). Here we describe a simple, efficient, repeatable and safe method to detect the presence of fungal structures using light microscopy.

The plant pathogenic fungus, Colletotrichum higginsianum is widely used to understand infection mechanisms, as it infects the model plant Arabidopsis thaliana. To determine the virulence of C. higginsianum, several methods have been developed, such as disease reaction scoring, lesion measurement, entry rate assays, and relative fungal biomass assays using real-time quantitative PCR. Although many studies have taken advantage of these methods, they have shortcomings in terms of objectivity, time, or cost. Here, we show a lesion area detection method applying ImageJ color thresholds to images of A. thaliana leaves infected by C. higginsianum. This method can automatically detect multiple lesions in a short time without the requirement for special equipment and measures lesion areas in a standardized way. This high throughput technique will aid better understanding of plant immunity and pathogenicity and contribute to reproducibility of assays.

Endocytosis is an intracellular trafficking pathway that occurs in nutrient uptake, signal transduction and reconstruction of cell polarity and is conserved in eukaryotic cells. In fungi, endocytosis plays crucial roles in the physiology of hyphal growth and pathogenicity. vidence for endocytosis in filamentous fungi is detected by the membrane-selective dyes FM4-64. Cells of a range of filamentous fungal species readily take up these dyes. However, the method for endocytosis detection has not been well established in Magnaporthe oryzae. Here, we provide a protocol for tracking endocytosis in Magnaporthe oryzae.

Symbiotic interactions between arbuscular mycorrhizal fungi (AMF) and plants are widespread among land plants and can be beneficial for both partners. The plant is provided with mineral nutrients such as nitrogen and phosphorous, whereas it provides carbon resources for the fungus in return. Due to the large economic and environmental impact, efficient characterization methods are required to monitor and quantify plant-AMF colonization. Existing methods, based on destructive sampling and elaborate root tissue analysis, are of limited value for high-throughput (HTP) screening. Here we describe a detailed protocol for the HTP quantification of blumenol derivatives in leaves by a simple extraction procedure and sensitive liquid chromatography mass spectrometry (LC/MS) analysis as accurate proxies of root AMF-associations in both model plants and economically relevant crops.

Blumeria graminis is a fungus that causes powdery mildews on grasses, such as barley. Investigations of this pathogen present many challenges due to its obligate biotrophic nature. This means that the fungus can only grow in the presence of a living host plant. B. graminis forms epiphytic mycelia on the plant surface and feeding organs (haustoria) inside the epidermal cells of the host plant. Therefore, it is difficult to separate the fungus from plant tissues. This protocol shows how to obtain different fungal structures from powdery mildew infected barley leaves. The epiphytic mycelia including conidia and conidiophores can be separated after immersing the infected leaves into 5% cellulose acetate dissolved in acetone, and peeling off the cellulose acetate membrane. Then, the haustoria are isolated from dissected epidermis after cellulase degradation of plant cell walls. The isolated haustoria remain intact with few plant impurities. The haustoria may be visualized by epifluorescence microscopy after staining with the chitin-specific dye WGA-Alexa Fluor 488. Finally, dissected material can be either processed immediately or kept at -80 °C for long-term storage for studies on gene expression and protein identification, for example by mass spectrometry.

Maize ear rot is a worldwide fungal disease mainly caused by Fusarium verticillioides and Fusarium graminearum. Maize planted in the field was inoculated with Fusarium verticillioides at the filling stage, 15 days after pollination. Two milliliters of spore suspension with a concentration of 5 x 10⁶/ml was injected into the middle of the top ear using pricking ear method to cause maize ear rot. The thirty days after inoculation was the most suitable time for phenotypic evaluation of Fusarium resistance.

Phytophthora sojae, the causal agent of soybean root and stem rot, is responsible for enormous economic losses in soybean production. P. sojae secrets various effectors to reprogram host immunity. The plant apoplastic space is a major battleground in plant-pathogen interactions. Here we describe a protocol for purification and isolation of secreted proteins from P. sojae, including precipitation of secreted proteins from P. sojae culture filtrate, chromatographic purification of the secreted proteins and analysis of the proteins by Mass spectrometry. With this protocol, it will be easier to identify potential apoplastic effectors in Phytophthora and will benefit our understanding of plant-microbe interactions.

Natural hosts for the fungal endophyte Epichloë festucae include Festuca rubra (fine fescue) and Festuca trachyphylla (hard fescue). Some strains also form stable associations with Lolium perenne (perennial ryegrass). L. perenne is a suitable host to study fungal endophyte–grass interactions, such as endophytic fungal growth within the plant and epiphyllous growth on the plant surface. Here we provide a detailed protocol based on work by, for artificial inoculation of E. festucae into L. perenne, and newly developed staining and visualization techniques for observing endophytic and epiphyllous hyphae and the expressorium, an appressorium-like structure used by the fungus to exit the plant. The staining method uses a combination of glucan binding aniline blue diammonium salt (AB) and chitin binding wheat germ agglutinin-conjugated Alexa Fluor^®488 -(WGA-AF488). This protocol will be a useful tool to study Epichloë-grass interactions, particularly the comparison of different Epichloë-grass associations, various endophyte-host developmental stages, as well as the analysis of mutant Epichloë strains.